Methods and compositions for the recombinant biosynthesis of fatty acids and esters

ABSTRACT

The present disclosure identifies methods and compositions for modifying photoautotrophic organisms, such that the organisms efficiently convert carbon dioxide and light into compounds such as esters and fatty acids. In certain embodiments, the compounds produced are secreted into the medium used to culture the organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of international applicationPCT/US/2009/035937, filed Mar. 3, 2009, which claims the benefit ofearlier filed U.S. Provisional Patent Application No. 61/121,532, filedDec. 10, 2008, U.S. Provisional Patent Application No. 61/033,411 filedMar. 3, 2008, and U.S. Provisional Application No. 61/033,402, filedMar. 3, 2008; this application also claims priority to U.S. ProvisionalApplication 61/353,145, filed Jun. 9, 2010. The disclosures of each ofthese applications are incorporated hereinby reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 23, 2010, is named16817US_sequencelisting.txt, lists 25 sequences, and is 93.6 kb in size.

FIELD OF THE INVENTION

The present disclosure relates to methods for conferring fatty acid andfatty acid ester-producing properties to a heterotrophic orphotoautotrophic host, such that the modified host can be used in thecommercial production of fuels and chemicals.

BACKGROUND OF THE INVENTION

Many existing photoautotrophic organisms (i.e., plants, algae, andphotosynthetic bacteria) are poorly suited for industrial bioprocessingand have therefore not demonstrated commercial viability. Such organismstypically have slow doubling times (3-72 hrs) compared to industrializedheterotrophic organisms such as Escherichia coli (20 minutes),reflective of low total productivities. A need exists, therefore, forengineered photosynthetic microbes which produce increased yields offatty acids and esters.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a method for producing fattyacid esters, comprising: (i) culturing an engineered photosyntheticmicroorganism in a culture medium, wherein said engineeredphotosynthetic microorganism comprises a recombinant thioesterase, arecombinant acyl-CoA synthetase, and a recombinant wax synthase; and(ii) exposing said engineered photosynthetic microorganism to light andcarbon dioxide, wherein said exposure results in the incorporation of analcohol into a fatty acid ester produced by said engineeredphotosynthetic microorganism. In a related embodiment, the engineeredphotosynthetic microorganism is an engineered cyanobacterium. In anotherrelated embodiment, at least one of said fatty acid esters produced bythe engineered cyanobacterium is selected from the group consisting of atetradecanoic acid ester, a hexadecanoic acid ester, a heptadecanoicacid ester, a Δ9-octadecenoic acid ester, and an octadecanoic acidester. In another related embodiment, the amount of said fatty acidesters produced by said engineered cyanobacterium is increased relativeto the amount of fatty acid produced by an otherwise identical celllacking said recombinant thioesterase, acyl-CoA synthetase or waxsynthase. In certain embodiments, the incorporated alcohol is anexogenously added alcohol selected from the group consisting ofmethanol, ethanol, propanol, isopropanol, butanol, hexanol,cyclohexanol, and isoamyl alcohol.

In another related embodiment, the esters produce by the engineeredcyanobacteria include a hexadecanoic acid ester and an octadecanoic acidester. In another related embodiment, the amount of hexadecanoic acidester produced is between 1.5 and 10 fold greater than the amount ofoctadecanoic acid ester. In yet another related embodiment, the amountof hexadecanoic acid ester produced is between 1.5 and 5 fold greaterthan the amount of octadecanoic acid ester produced. In yet anotherrelated embodiment, at least 50% of the esters produced by saidengineered cyanobacterium are hexadecanoic acid esters. In yet anotherrelated embodiment, between 65% and 85% of the esters produced by saidengineered cyanobacterium are hexadecanoic acid esters.

In a related embodiment of the method for producing fatty acid estersdescribed above, the exogenously alcohol is butanol and fatty acidybutyl esters are produced. In yet another related embodiment, the yieldof fatty acid butyl esters is at least 5% dry cell weight. In yetanother related embodiment, the yield of fatty acid butyl esters is atleast 10% dry cell weight. In yet another related embodiment,exogenously added butanol is present in said culture at concentrationsbetween 0.01 and 0.2% (vol/vol). In yet another related embodiment, theconcentration of exogenously added butantol is about 0.05 to 0.075%(vol/vol).

In another related embodiment of the method for producing fatty acidesters described above, the exogenously added alcohol is ethanol. In yetanother related embodiment, the yield of ethyl esters is at least 1% drycell weight.

In another related embodiment of the method for producing fatty acidesters described above, the exogenously added alcohol is methanol. Inyet another related embodiment, the yield of methyl esters is at least0.01% dry cell weight.

In another related embodiment, said engineered cyanobacterium furthercomprises a recombinant resistance nodulation cell division type(“RND-type”) transporter, e.g., a TolC-AcrAB transporter. In anotherrelated embodiment, the expression of TolC is controlled by a promoterseparate from the promoter that controls expression of AcrAB. In anotherrelated embodiment, the genes encoding the recombinant transporter areencoded by a plasmid. In another related embodiment, the fatty acidesters are secreted into the culture medium at increased levels relativeto an otherwise identical cyanobacterium lacking the recombinanttransporter.

In certain embodiments of the methods for producing fatty acid estersdescribed above, the recombinant thioesterase, wax synthase, andacyl-CoA synthetase are expressed as an operon under the control of asingle promoter. In certain embodiments, the single promoter is aninducible promoter. In other embodiments of the methods described above,the expression of at least two of the genes selected from the groupconsisting of a recombinant thioesterase, wax synthase, and acyl-CoAsynthetase is under the control of different promoters. One or more ofthe promoters can be an inducible promoter. In related embodiments, atleast one of said recombinant genes is encoded on a plasmid. In yetother related embodiments, at least one of said recombinant genes isintegrated into the chromosome of the engineered cyanobacteria. In yetother related embodiments, at least one of said recombinant genes is agene that is native to the engineered cyanobacteria, but whoseexpression is controlled by a recombinant promoter. In yet other relatedembodiments, one or more promoters are selected from the groupconsisting of a cI promoter, a cpcB promoter, a lacI-Ptrc promoter, anEM7 promoter, an PaphII promoter, a NirA-type promoter, a PnrsApromoter, or a PnrsB promoter.

In another embodiment, the invention provides a method for producingfatty acid esters, comprising: (i) culturing an engineeredcyanobacterium in a culture medium, wherein said engineeredcyanobacterium comprises a recombinant acyl-CoA synthetase and arecombinant wax synthase; and (ii) exposing said engineeredcyanobacterium to light and carbon dioxide, wherein said exposureresults in the conversion of an alcohol by said engineeredcynanobacterium into fatty acid esters, wherein at least one of saidfatty acid esters is selected from the group consisting of atetradecanoic acid ester, a hexadecanoic acid ester, a heptadecanoicacid ester, a Δ9-octadecenoic acid ester, and an octadecanoic acidester, wherein the amount of said fatty acid esters produced by saidengineered cyanobacterium is increased relative to the amount of fattyacid produced by an otherwise identical cell lacking said recombinantacyl-CoA synthetase or wax synthase. In a related embodiment, thealcohol is an exogenously added alcohol selected from the groupconsisting of methanol, ethanol, propanol, isopropanol, butanol,hexanol, cyclohexanol, and isoamyl alcohol.

In another embodiment, the invention provides a method for producing afatty acid ester, comprising: (i) culturing an engineered cyanobacteriumin a culture medium, wherein said engineered cyanobacterium comprises arecombinant RND-type transporter; and (ii) exposing said engineeredcyanobacterium to light and carbon dioxide, wherein said exposureresults in the production of a fatty acid ester by said engineeredcyanobacterium, and wherein said RND-type transporter secretes saidfatty acid ester into said culture medium. In a related embodiment, saidRND-type transporter is a TolC-AcrAB transporter.

In an embodiment related to the methods described above, the inventionfurther comprises isolating said fatty acid ester from said engineeredcyanobacterium or said culture medium.

In another embodiment, the invention also provides an engineeredcyanobacterium, wherein said cyanobacterium comprises a recombinantthioesterase, a recombinant acyl-CoA synthetase, and a recombinant waxsynthase. In certain embodiments, the engineered cyanobacteriumadditionally comprises a recombinant RND-type transporter, e.g., aTolC-AcrAB transporter.

In a related embodiment, at least one of said recombinant enzymes isheterologous with respect to said engineered cyanobacterium. In anotherembodiment, said cyanobacterium does not synthesize fatty acid esters inthe absence of the expression of one or both of the recombinant enzymes.In another embodiment, at least one of said recombinant enzymes is notheterologous to said engineered cyanobacterium.

In yet another related embodiment, the recombinant thioesterase,acyl-CoA synthetase and wax synthase are selected from the enzymeslisted in Table 3A, Table 3B and Table 3C, respectively. In yet anotherrelated embodiment, the recombinant thioesterase has an amino acidsequence that is identical to SEQ ID NO: 1. In yet another relatedembodiment, the recombinant thioesterase has an amino acid sequence thatis at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,or at least 99% identical to SEQ ID NO: 1. In yet another relatedembodiment, the recombinant acyl-CoA synthetase is identical to SEQ IDNO:2. In yet another related embodiment, the recombinant acyl-CoAsynthetase has an amino acid sequence at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2. In yet another related embodiment, recombinant wax synthase isidentical to SEQ ID NO: 3. In yet another related embodiment, therecombinant wax synthase has an amino acid sequence is at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 3. In yet another related embodiment, therecombinant TolC transporter amino acid sequence is identical to SEQ IDNO: 7. In yet another related embodiment, the recombinant TolCtransporter has an amino acid sequence at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 7. In yet another related embodiment, the recombinant AcrA aminoacid sequence is identical to SEQ ID NO: 8. In yet another relatedembodiment, the recombinant AcrA amino acid sequence is at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 8. In yet another related embodiment, therecombinant AcrB amino acid sequence is identical to SEQ ID NO: 9. Inyet another related embodiment, the recombinant AcrB amino acid sequenceis at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,or at least 99% identical to SEQ ID NO: 9.

In related embodiments of the above-described embodiments, an engineeredphotosynthetic microorganism other than a cyanobacterium can be used. Inother related embodiments, a thermophilic cyanobacterium can be used.

In another embodiment, the invention provides a methods and compositionsfor producing fatty acids using an engineered photosyntheticmicroorganism. For example, in one embodiment, the invention provides amethod for producing fatty acids, comprising: (a) culturing anengineered photosynthetic microorganism, wherein said engineeredphotosynthetic microorganism comprises a modification which reduces theexpression of said microorganism's endogenous acyl-ACP synthetase; and(b) exposing said engineered photosynthetic microorganism to light andcarbon dioxide, wherein said exposure results in the production of fattyacids by said engineered cyanobacterium, wherein the amount of fattyacids produced is increased relative to the amount of fatty acidsproduced by an otherwise identical microorganism lacking saidmodification. In a related embodiment, the engineered microorganism is athermophile. In another related embodiment, the engineered microorganismis a cyanobacterium. In yet another related embodiment, the engineeredmicroorganism is a thermophilic cyanobacterium. In yet another relatedembodiment, the engineered microorganism is Thermosynechococcuselongatus BP-1. In yet another related embodiment of the method forproducing fatty acids, the modification is a knock-out or deletion ofthe gene encoding said endogenous acyl-ACP synthetase. In yet anotherrelated embodiment, the gene encoding said acyl-ACP synthetase is theacyl-ACP synthetase or aas gene, e.g., GenBank accession numberNP_(—)682091.1. In yet another related embodiment, the increase in fattyacid production is at least a 2 fold increase. In yet another relatedembodiment, the increase in fatty acid production is between 2 and 4.5fold. In yet another related embodiment, the increase in fatty acidproduction includes an increase in fatty acids secreted into a culturemedia. In yet another related embodiment, most of said increase in fattyacid production arises from the increased production of myristic andoleic acid. In yet another related embodiment of the method forproducing fatty acids, the engineereed photosynthetic microorganismfurther comprises a TolC-AcrAB transporter.

In another embodiment, the invention provides an engineeredphotosynthetic microorganism, wherein said microorganism comprises adeletion or knock-out of an endogenous gene encoding a acyl-ACPsynthetase or long-chain fatty acid ligase. In a related embodiment,engineered photosynthetic microorganism is a thermophile. In yet anotherrelated embodiment, the engineered photosynthetic microorganism is acyanobacterium or a thermophilic cyanobacterium. In yet another relatedembodiment, the cyanobacterium is Thermosynechococcus elongatus BP-1. Inyet another related embodiment, the acyl-ACP synthetase is the aas geneof the thermophilic cyanobacterium, e.g., GenBank accession numberNP_(—)682091.1. In yet another related embodiment, the engineereedphotosynthetic microorganism further comprises a TolC-AcrAB transporter.

In yet another embodiment, the invention provides an engineeredcyanbacterial strain selected from the group consisting of JCC723,JCC803, JCC1215, JCC803, JCC1132, and JCC1585. In yet anotherembodiment, the invention provides an engineered cyanobacterial strainselected from the group consisting of the engineered Synechococcus sp.PCC7002 strains JCC1648 (Δaas tesA, with tesA under control of P(nir07)on pAQ4), JCC1704 (Δaas fatB, with fatB inserted at aquI under thecontrol of P(nir07)), JCC1705 (Δaas fatB1, with fatB1 inserted at aquIunder the control of P(nir07)), JCC1706 (Δaas fatB2 with fatB2 insertedat aquI under the control of P(nir07)), JCC1751 (Δaas tesA, with tesAunder control of P(nir07) on pAQ3), and JCC1755 (Δaas fatB_mat, withfatB_mat under control of P(nir07) on pAQ3). In yet another embodiment,the invention provides the engineered cyanobacterial strain JCC1862(Thermosynechococcus elongatus BP-1 kan^(R) Δaas).

These and other embodiments of the invention are further described inthe Figures, Description, Examples and Claims, herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a GC/MS chromatogram overlay comparing cell pelletextracts of JCC803 incubated with either methanol (top trace) or ethanol(bottom traces). The peaks due to methyl esters (MEs) or ethyl esters(EEs) are labeled.

FIG. 2 shows three stacked GC/FID chromatograms comparing cell pelletextracts of the indicated cyanobacterial strains when cultured in thepresence of ethanol. The interval between tick marks on the FID responseaxis is 20,000.

FIG. 3 depicts stacks of GC/FID chromatograms comparing cell pelletextracts of JCC803 cultures incubated with different alcohols (indicatedon respective chromatograms). Numbers indicate the respective fatty acidester corresponding to the alcohol added (1=myristate; 2=palmitate;3=oleate; 4=stearate). EA=ethyl arachidate. The interval between tickmarks on the FID response axis is 400,000.

FIG. 4 depicts a GC/chromatogram of a cell pellet extract from a JCC803culture incubated with ethanol. 1=ethyl myristate; 2=ethyl palmitoleate;3=ethyl palmitate; 4=ethyl margarate; 5=ethyl oleate; 6=ethyl stearate.

FIG. 5 depicts a GC/chromatogram of a cell pellet extract from a JCC803culture incubated with butanol. 1=butyl myristate, 2=butyl palmitoleate,3=butyl palmitate, 4=butyl margarate, 5=butyl oleate, 6=butyl stearate.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall include theplural and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, biochemistry,enzymology, molecular and cellular biology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer,Introduction to Glycobiology, Oxford Univ. Press (2003); WorthingtonEnzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbookof Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbookof Biochemistry: Section A Proteins, Vol II, CRC Press (1976);Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

All publications, patents and other references mentioned herein arehereby incorporated by reference in their entireties.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinternucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation.

Unless otherwise indicated, and as an example for all sequencesdescribed herein under the general format “SEQ ID NO:”, “nucleic acidcomprising SEQ ID NO:1” refers to a nucleic acid, at least a portion ofwhich has either (i) the sequence of SEQ ID NO:1, or (ii) a sequencecomplementary to SEQ ID NO:1. The choice between the two is dictated bythe context. For instance, if the nucleic acid is used as a probe, thechoice between the two is dictated by the requirement that the probe becomplementary to the desired target.

An “isolated” RNA, DNA or a mixed polymer is one which is substantiallyseparated from other cellular components that naturally accompany thenative polynucleotide in its natural host cell, e.g., ribosomes,polymerases and genomic sequences with which it is naturally associated.

As used herein, an “isolated” organic molecule (e.g., a fatty acid or afatty acid ester) is one which is substantially separated from thecellular components (membrane lipids, chromosomes, proteins) of the hostcell from which it originated, or from the medium in which the host cellwas cultured. The term does not require that the biomolecule has beenseparated from all other chemicals, although certain isolatedbiomolecules may be purified to near homogeneity.

The term “recombinant” refers to a biomolecule, e.g., a gene or protein,that (1) has been removed from its naturally occurring environment, (2)is not associated with all or a portion of a polynucleotide in which thegene is found in nature, (3) is operatively linked to a polynucleotidewhich it is not linked to in nature, or (4) does not occur in nature.The term “recombinant” can be used in reference to cloned DNA isolates,chemically synthesized polynucleotide analogs, or polynucleotide analogsthat are biologically synthesized by heterologous systems, as well asproteins and/or mRNAs encoded by such nucleic acids.

As used herein, an endogenous nucleic acid sequence in the genome of anorganism (or the encoded protein product of that sequence) is deemed“recombinant” herein if a heterologous sequence is placed adjacent tothe endogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. In this context, aheterologous sequence is a sequence that is not naturally adjacent tothe endogenous nucleic acid sequence, whether or not the heterologoussequence is itself endogenous (originating from the same host cell orprogeny thereof) or exogenous (originating from a different host cell orprogeny thereof). By way of example, a promoter sequence can besubstituted (e.g., by homologous recombination) for the native promoterof a gene in the genome of a host cell, such that this gene has analtered expression pattern. This gene would now become “recombinant”because it is separated from at least some of the sequences thatnaturally flank it.

A nucleic acid is also considered “recombinant” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “recombinant” if it contains an insertion, deletion or apoint mutation introduced artificially, e.g., by human intervention. A“recombinant nucleic acid” also includes a nucleic acid integrated intoa host cell chromosome at a heterologous site and a nucleic acidconstruct present as an episome.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence. The term “degenerate oligonucleotide” or “degenerate primer”is used to signify an oligonucleotide capable of hybridizing with targetnucleic acid sequences that are not necessarily identical in sequencebut that are homologous to one another within one or more particularsegments.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. Pearson, MethodsEnzymol. 183:63-98 (1990) (hereby incorporated by reference in itsentirety). For instance, percent sequence identity between nucleic acidsequences can be determined using FASTA with its default parameters (aword size of 6 and the NOPAM factor for the scoring matrix) or using Gapwith its default parameters as provided in GCG Version 6.1, hereinincorporated by reference. Alternatively, sequences can be comparedusing the computer program, BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 76%, 80%, 85%, preferablyat least about 90%, and more preferably at least about 95%, 96%, 97%,98% or 99% of the nucleotide bases, as measured by any well-knownalgorithm of sequence identity, such as FASTA, BLAST or Gap, asdiscussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference.For purposes herein, “stringent conditions” are defined for solutionphase hybridization as aqueous hybridization (i.e., free of formamide)in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1%SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1%SDS at 65° C. for 20 minutes. It will be appreciated by the skilledworker that hybridization at 65° C. will occur at different ratesdepending on a number of factors including the length and percentidentity of the sequences which are hybridizing.

The nucleic acids (also referred to as polynucleotides) of this presentinvention may include both sense and antisense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Theymay be modified chemically or biochemically or may contain non-naturalor derivatized nucleotide bases, as will be readily appreciated by thoseof skill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.) Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule. Other modifications can include, for example, analogs in whichthe ribose ring contains a bridging moiety or other structure such asthe modifications found in “locked” nucleic acids.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989)and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and“oligonucleotide-directed mutagenesis” (a process which enables thegeneration of site-specific mutations in any cloned DNA segment ofinterest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57(1988)).

The term “attenuate” as used herein generally refers to a functionaldeletion, including a mutation, partial or complete deletion, insertion,or other variation made to a gene sequence or a sequence controlling thetranscription of a gene sequence, which reduces or inhibits productionof the gene product, or renders the gene product non-functional. In someinstances a functional deletion is described as a knockout mutation.Attenuation also includes amino acid sequence changes by altering thenucleic acid sequence, placing the gene under the control of a lessactive promoter, down-regulation, expressing interfering RNA, ribozymesor antisense sequences that target the gene of interest, or through anyother technique known in the art. In one example, the sensitivity of aparticular enzyme to feedback inhibition or inhibition caused by acomposition that is not a product or a reactant (non-pathway specificfeedback) is lessened such that the enzyme activity is not impacted bythe presence of a compound. In other instances, an enzyme that has beenaltered to be less active can be referred to as attenuated.

Deletion: The removal of one or more nucleotides from a nucleic acidmolecule or one or more amino acids from a protein, the regions oneither side being joined together.

Knock-out: A gene whose level of expression or activity has been reducedto zero. In some examples, a gene is knocked-out via deletion of some orall of its coding sequence. In other examples, a gene is knocked-out viaintroduction of one or more nucleotides into its open reading frame,which results in translation of a non-sense or otherwise non-functionalprotein product.

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which generally refersto a circular double stranded DNA loop into which additional DNAsegments may be ligated, but also includes linear double-strandedmolecules such as those resulting from amplification by the polymerasechain reaction (PCR) or from treatment of a circular plasmid with arestriction enzyme. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certainpreferred vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply “expression vectors”).

“Operatively linked” or “operably linked” expression control sequencesrefers to a linkage in which the expression control sequence iscontiguous with the gene of interest to control the gene of interest, aswell as expression control sequences that act in trans or at a distanceto control the gene of interest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

Promoters useful for expressing the recombinant genes described hereininclude both constitutive and inducible/repressible promoters. Examplesof inducible/repressible promoters include nickel-inducible promoters(e.g., PnrsA, PnrsB; see, e.g., Lopez-Mauy et al., Cell (2002) v.43:247-256, incorporated by reference herein) and urea repressiblepromoters such as PnirA (described in, e.g., Qi et al., Applied andEnvironmental Microbiology (2005) v. 71: 5678-5684, incorporated byreference herein). In other embodiments, a PaphII and/or a lacIq-Ptrcpromoter can used to control expression. Where multiple recombinantgenes are expressed in an engineered cyanobacteria of the invention, thedifferent genes can be controlled by different promoters or by identicalpromoters in separate operons, or the expression of two or more genesmay be controlled by a single promoter as part of an operon.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally-occurring proteins, and fragments, mutants, derivativesand analogs thereof. A polypeptide may be monomeric or polymeric.Further, a polypeptide may comprise a number of different domains eachof which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) exists in a purity not found in nature, wherepurity can be adjudged with respect to the presence of other cellularmaterial (e.g., is free of other proteins from the same species) (3) isexpressed by a cell from a different species, or (4) does not occur innature (e.g., it is a fragment of a polypeptide found in nature or itincludes amino acid analogs or derivatives not found in nature orlinkages other than standard peptide bonds). Thus, a polypeptide that ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A polypeptide or protein may alsobe rendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.As thus defined, “isolated” does not necessarily require that theprotein, polypeptide, peptide or oligopeptide so described has beenphysically removed from its native environment.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has a deletion, e.g., an amino-terminal and/or carboxy-terminaldeletion compared to a full-length polypeptide. In a preferredembodiment, the polypeptide fragment is a contiguous sequence in whichthe amino acid sequence of the fragment is identical to thecorresponding positions in the naturally-occurring sequence. Fragmentstypically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferablyat least 12, 14, 16 or 18 amino acids long, more preferably at least 20amino acids long, more preferably at least 25, 30, 35, 40 or 45, aminoacids, even more preferably at least 50 or 60 amino acids long, and evenmore preferably at least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those skilledin the art. A variety of methods for labeling polypeptides and ofsubstituents or labels useful for such purposes are well known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well known in the art. See, e.g.,Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992, and Supplements to 2002) (herebyincorporated by reference).

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusions that include theentirety of the proteins of the present invention have particularutility. The heterologous polypeptide included within the fusion proteinof the present invention is at least 6 amino acids in length, often atleast 8 amino acids in length, and usefully at least 15, 20, and 25amino acids in length. Fusions that include larger polypeptides, such asan IgG Fc region, and even entire proteins, such as the greenfluorescent protein (“GFP”) chromophore-containing proteins, haveparticular utility. Fusion proteins can be produced recombinantly byconstructing a nucleic acid sequence which encodes the polypeptide or afragment thereof in frame with a nucleic acid sequence encoding adifferent protein or peptide and then expressing the fusion protein.Alternatively, a fusion protein can be produced chemically bycrosslinking the polypeptide or a fragment thereof to another protein.

As used herein, the term “antibody” refers to a polypeptide, at least aportion of which is encoded by at least one immunoglobulin gene, orfragment thereof, and that can bind specifically to a desired targetmolecule. The term includes naturally-occurring forms, as well asfragments and derivatives.

Fragments within the scope of the term “antibody” include those producedby digestion with various proteases, those produced by chemical cleavageand/or chemical dissociation and those produced recombinantly, so longas the fragment remains capable of specific binding to a targetmolecule. Among such fragments are Fab, Fab′, Fv, F(ab′).sub.2, andsingle chain Fv (scFv) fragments.

Derivatives within the scope of the term include antibodies (orfragments thereof) that have been modified in sequence, but remaincapable of specific binding to a target molecule, including:interspecies chimeric and humanized antibodies; antibody fusions;heteromeric antibody complexes and antibody fusions, such as diabodies(bispecific antibodies), single-chain diabodies, and intrabodies (see,e.g., Intracellular Antibodies: Research and Disease Applications,(Marasco, ed., Springer-Verlag New York, Inc., 1998), the disclosure ofwhich is incorporated herein by reference in its entirety).

As used herein, antibodies can be produced by any known technique,including harvest from cell culture of native B lymphocytes, harvestfrom culture of hybridomas, recombinant expression systems and phagedisplay.

The term “non-peptide analog” refers to a compound with properties thatare analogous to those of a reference polypeptide. A non-peptidecompound may also be termed a “peptide mimetic” or a “peptidomimetic.”See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford UniversityPress (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: AHandbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—APractical Textbook, Springer Verlag (1993); Synthetic Peptides: A UsersGuide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med.Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veberand Freidinger, Trends Neurosci., 8:392-396 (1985); and references sitedin each of the above, which are incorporated herein by reference. Suchcompounds are often developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar to usefulpeptides of the present invention may be used to produce an equivalenteffect and are therefore envisioned to be part of the present invention.

A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild-type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occurringprotein.

A mutein has at least 85% overall sequence homology to its wild-typecounterpart. Even more preferred are muteins having at least 90% overallsequence homology to the wild-type protein.

In an even more preferred embodiment, a mutein exhibits at least 95%sequence identity, even more preferably 98%, even more preferably 99%and even more preferably 99.9% overall sequence identity.

Sequence homology may be measured by any common sequence analysisalgorithm, such as Gap or Bestfit.

Amino acid substitutions can include those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2^(nd) ed.1991), which is incorporated herein by reference. Stereoisomers (e.g.,D-amino acids) of the twenty conventional amino acids, unnatural aminoacids such as α-, α-disubstituted amino acids, N-alkyl amino acids, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand end corresponds to the amino terminal end and the right-handend corresponds to the carboxy-terminal end, in accordance with standardusage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences.) As used herein, homology between tworegions of amino acid sequence (especially with respect to predictedstructural similarities) is interpreted as implying similarity infunction.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art. See,e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (hereinincorporated by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using a measure of homology assignedto various substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild-type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a particular polypeptide sequenceto a database containing a large number of sequences from differentorganisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

Preferred parameters for BLASTp are: Expectation value: 10 (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

Preferred parameters for BLASTp are: Expectation value: 10 (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62. The length of polypeptide sequences compared for homologywill generally be at least about 16 amino acid residues, usually atleast about 20 residues, more usually at least about 24 residues,typically at least about 28 residues, and preferably more than about 35residues. When searching a database containing sequences from a largenumber of different organisms, it is preferable to compare amino acidsequences. Database searching using amino acid sequences can be measuredby algorithms other than blastp known in the art. For instance,polypeptide sequences can be compared using FASTA, a program in GCGVersion 6.1. FASTA provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences.Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated by referenceherein). For example, percent sequence identity between amino acidsequences can be determined using FASTA with its default parameters (aword size of 2 and the PAM250 scoring matrix), as provided in GCGVersion 6.1, herein incorporated by reference.

“Specific binding” refers to the ability of two molecules to bind toeach other in preference to binding to other molecules in theenvironment. Typically, “specific binding” discriminates overadventitious binding in a reaction by at least two-fold, more typicallyby at least 10-fold, often at least 100-fold. Typically, the affinity oravidity of a specific binding reaction, as quantified by a dissociationconstant, is about 10⁻⁷ M or stronger (e.g., about 10⁻⁸ M, 10⁻⁹ M oreven stronger).

“Percent dry cell weight” refers to a production measurement of estersof fatty acids or fatty acids obtained as follows: a defined volume ofculture is centrifuged to pellet the cells. Cells are washed thendewetted by at least one cycle of microcentrifugation and aspiration.Cell pellets are lyophilized overnight, and the tube containing the drycell mass is weighed again such that the mass of the cell pellet can becalculated within ±0.1 mg. At the same time cells are processed for drycell weight determination, a second sample of the culture in question isharvested, washed, and dewetted. The resulting cell pellet,corresponding to 1-3 mg of dry cell weight, is then extracted byvortexing in approximately 1 ml acetone plus butylated hydroxytolune(BHT) as antioxidant and an internal standard, e.g., ethyl arachidate.Cell debris is then pelleted by centrifugation and the supernatant(extractant) is taken for analysis by GC. For accurate quantitation ofthe molecules, flame ionization detection (FID) was used as opposed toMS total ion count. The concentrations of the esters or fatty acids inthe biological extracts were calculated using calibration relationshipsbetween GC-FID peak area and known concentrations of authenticstandards. Knowing the volume of the extractant, the resultingconcentrations of the products in the extractant, and the dry cellweight of the cell pellet extracted, the percentage of dry cell weightthat comprised the esters or fatty acids can be determined.

The term “region” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, an Igdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

“Carbon-based Products of Interest” include alcohols such as ethanol,propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, waxesters; hydrocarbons and alkanes such as propane, octane, diesel, JetPropellant 8 (JP8); polymers such as terephthalate, 1,3-propanediol,1,4-butanediol, polyols, Polyhydroxyalkanoates (PHA),poly-beta-hydroxybutyrate (PHB), acrylate, adipic acid, ε-caprolactone,isoprene, caprolactam, rubber; commodity chemicals such as lactate,Docosahexaenoic acid (DHA), 3-hydroxypropionate, γ-valerolactone,lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbicacid, isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate,1,3-butadiene, ethylene, propylene, succinate, citrate, citric acid,glutamate, malate, 3-hydroxypropionic acid (HPA), lactic acid, THF,gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid,levulinic acid, acrylic acid, malonic acid; specialty chemicals such ascarotenoids, isoprenoids, itaconic acid; pharmaceuticals andpharmaceutical intermediates such as 7-aminodeacetoxycephalosporanicacid (7-ADCA)/cephalosporin, erythromycin, polyketides, statins,paclitaxel, docetaxel, terpenes, peptides, steroids, omega fatty acidsand other such suitable products of interest. Such products are usefulin the context of biofuels, industrial and specialty chemicals, asintermediates used to make additional products, such as nutritionalsupplements, neutraceuticals, polymers, paraffin replacements, personalcare products and pharmaceuticals.

Biofuel: A biofuel refers to any fuel that derives from a biologicalsource. Biofuel can refer to one or more hydrocarbons, one or morealcohols, one or more fatty esters or a mixture thereof.

The term “hydrocarbon” generally refers to a chemical compound thatconsists of the elements carbon (C), hydrogen (H) and optionally oxygen(O). There are essentially three types of hydrocarbons, e.g., aromatichydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons suchas alkenes, alkynes, and dienes. The term also includes fuels, biofuels,plastics, waxes, solvents and oils. Hydrocarbons encompass biofuels, aswell as plastics, waxes, solvents and oils. A “fatty acid” is acarboxylic acid with a long unbranched aliphatic tail (chain), which iseither saturated or unsaturated. Most naturally occurring fatty acidshave a chain of four to 28 carbons.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this present invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Nucleic Acid Sequences

Esters are chemical compounds with the basic formula:

where R and R′ denote any alkyl or aryl group. In one embodiment, theinvention provides one or more isolated or recombinant nucleic acidsencoding one or more genes which, when recombinantly expressed in aphotosynthetic microorganism, catalyze the synthesis of esters by themicroorganism. The first gene is a thioesterase, which catalyzes thesynthesis of fatty acids from an acyl-Acyl Carrier Protein (“acyl-ACP”)molecule. The second gene is an acyl-CoA synthetase, which synthesizesfatty acyl-CoA from a fatty acid. The third gene is a wax synthase,which synthesizes esters from a fatty acyl-CoA molecule and an alcohol(e.g., methanol, ethanol, proponal, butanol, etc.). In certain relatedembodiments, additional genes expressing a recombinant resistancenodulation cell division type (“RND-type”) transporter such asTolC/AcrAB are also recombinantly expressed to facilitate the transportof ethyl esters outside of the engineered photosynthetic cell and intothe culture medium.

Accordingly, the present invention provides isolated nucleic acidmolecules for genes encoding thioesterase, acyl-CoA synthetases and waxsynthase enzymes, and variants thereof. An exemplary full-lengthexpression optimzed nucleic acid sequence for a gene encoding athioesterase is presented as SEQ ID NO: 4. The corresponding amino acidsequences is presented as SEQ ID NO: 1. Additional genes encodingthioesterases are presented in Table 3A. An exemplary full-lengthexpression-optimized nucleic acid sequence for a gene encoding anacyl-CoA synthetase is presented as SEQ ID NO: 5, and the correspondingamino acid sequence is presented as SEQ ID NOs: 2. Additional genesncoding acyl-CoA synthetases are presented in Table 3B. An exemplaryfull-length expression-optimized nucleic acid sequence for a geneencoding an acyl-CoA synthetase is presented as SEQ ID NO: 6, and thecorresponding amino acid sequence is presented as SEQ ID NOs: 3.Additional genes encoding acyl-CoA synthetases are presented in Table3C.

One skilled in the art will recognize that the redundancy of the geneticcode will allow many other nucleic acid sequences to encode theidentical enzymes. The sequences of the nucleic acids disclosed hereincan be optimized as needed to yield the desired expression levels in aparticular photosynthetic microorganism. Such a nucleic acid sequencecan have 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or even higheridentity to the native gene sequence.

In another embodiment, the nucleic acid molecule of the presentinvention encodes a polypeptide having the amino acid sequence of SEQ IDNO:1, 2, 3, 7, 8, or 9. Preferably, the nucleic acid molecule of thepresent invention encodes a polypeptide sequence of at least 50%, 60,70%, 80%, 85%, 90% or 95% identity to SEQ ID NO:1, 2, 3, 7, 8 or 9 andthe identity can even more preferably be 96%, 97%, 98%, 99%, 99.9% oreven higher.

The present invention also provides nucleic acid molecules thathybridize under stringent conditions to the above-described nucleic acidmolecules. As defined above, and as is well known in the art, stringenthybridizations are performed at about 25° C. below the thermal meltingpoint (T_(m)) for the specific DNA hybrid under a particular set ofconditions, where the T_(m) is the temperature at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Stringentwashing is performed at temperatures about 5° C. lower than the T_(m)for the specific DNA hybrid under a particular set of conditions.

Nucleic acid molecules comprising a fragment of any one of theabove-described nucleic acid sequences are also provided. Thesefragments preferably contain at least 20 contiguous nucleotides. Morepreferably the fragments of the nucleic acid sequences contain at least25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguousnucleotides.

The nucleic acid sequence fragments of the present invention displayutility in a variety of systems and methods. For example, the fragmentsmay be used as probes in various hybridization techniques. Depending onthe method, the target nucleic acid sequences may be either DNA or RNA.The target nucleic acid sequences may be fractionated (e.g., by gelelectrophoresis) prior to the hybridization, or the hybridization may beperformed on samples in situ. One of skill in the art will appreciatethat nucleic acid probes of known sequence find utility in determiningchromosomal structure (e.g., by Southern blotting) and in measuring geneexpression (e.g., by Northern blotting). In such experiments, thesequence fragments are preferably detectably labeled, so that theirspecific hydridization to target sequences can be detected andoptionally quantified. One of skill in the art will appreciate that thenucleic acid fragments of the present invention may be used in a widevariety of blotting techniques not specifically described herein.

It should also be appreciated that the nucleic acid sequence fragmentsdisclosed herein also find utility as probes when immobilized onmicroarrays. Methods for creating microarrays by deposition and fixationof nucleic acids onto support substrates are well known in the art.Reviewed in DNA Microarrays: A Practical Approach (Practical ApproachSeries), Schena (ed.), Oxford University Press (1999) (ISBN:0199637768); Nature Genet. 21(1) (suppl):1-60 (1999); MicroarrayBiochip: Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of which are incorporated herein by reference in theirentireties. Analysis of, for example, gene expression using microarrayscomprising nucleic acid sequence fragments, such as the nucleic acidsequence fragments disclosed herein, is a well-established utility forsequence fragments in the field of cell and molecular biology. Otheruses for sequence fragments immobilized on microarrays are described inGerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger,Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A PracticalApproach (Practical Approach Series), Schena (ed.), Oxford UniversityPress (1999) (ISBN: 0199637768); Nature Genet. 21(1) (suppl):1-60(1999); Microarray Biochip: Tools and Technology, Schena (ed.), EatonPublishing Company/BioTechniques Books Division (2000) (ISBN:1881299376), the disclosure of each of which is incorporated herein byreference in its entirety.

As is well known in the art, enzyme activities can be measured invarious ways. For example, the pyrophosphorolysis of OMP may be followedspectroscopically (Grubmeyer et al., (1993) J. Biol. Chem.268:20299-20304). Alternatively, the activity of the enzyme can befollowed using chromatographic techniques, such as by high performanceliquid chromatography (Chung and Sloan, (1986) J. Chromatogr.371:71-81). As another alternative the activity can be indirectlymeasured by determining the levels of product made from the enzymeactivity. These levels can be measured with techniques including aqueouschloroform/methanol extraction as known and described in the art (Cf. M.Kates (1986) Techniques of Lipidology; Isolation, analysis andidentification of Lipids. Elsevier Science Publishers, New York (ISBN:0444807322)). More modern techniques include using gas chromatographylinked to mass spectrometry (Niessen, W. M. A. (2001). Current practiceof gas chromatography—mass spectrometry. New York, N.Y.: Marcel Dekker.(ISBN: 0824704738)). Additional modern techniques for identification ofrecombinant protein activity and products including liquidchromatography-mass spectrometry (LCMS), high performance liquidchromatography (HPLC), capillary electrophoresis, Matrix-Assisted LaserDesorption Ionization time of flight-mass spectrometry (MALDI-TOF MS),nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy,viscometry (Knothe, G (1997) Am. Chem. Soc. Symp. Series, 666: 172-208),titration for determining free fatty acids (Komers (1997) Fett/Lipid,99(2): 52-54), enzymatic methods (Bailer (1991) Fresenius J. Anal. Chem.340(3): 186), physical property-based methods, wet chemical methods,etc. can be used to analyze the levels and the identity of the productproduced by the organisms of the present invention. Other methods andtechniques may also be suitable for the measurement of enzyme activity,as would be known by one of skill in the art.

Vectors

Also provided are vectors, including expression vectors, which comprisethe above nucleic acid molecules of the present invention, as describedfurther herein. In a first embodiment, the vectors include the isolatednucleic acid molecules described above. In an alternative embodiment,the vectors of the present invention include the above-described nucleicacid molecules operably linked to one or more expression controlsequences. The vectors of the instant invention may thus be used toexpress a thioesterase, an acyl-CoA synthease, and/or a wax synthase,contributing to the synthesis of esters by the cell.

In a related embodiment, vectors may include nucleic acid moleculesencoding an RND-type transporter such as TolC/AcrAB to facilitate theextracellular transport of esters. Exemplary vectors of the inventioninclude any of the vectors expressing a thioesterase, an acyl-CoAsynthease, wax synthase, and/or TolC/AcrAB transporter disclosed here,e.g., pJB532, pJB634, pJB578 and pJB1074. The invention also providesother vectors such as pJB161 which are capable of receiving nucleic acidsequences of the invention. Vectors such as pJB161 comprise sequenceswhich are homologous with sequences that are present in plasmids whichare endogenous to certain photosynthetic microorganisms (e.g., plasmidspAQ7 or pAQ1 of certain Synechococcus species). Recombination betweenpJB161 and the endogenous plasmids in vivo yield engineered microbesexpressing the genes of interest from their endogenous plasmids.Alternatively, vectors can be engineered to recombine with the host cellchromosome, or the vector can be engineered to replicate and expressgenes of interest independent of the host cell chromosome or any of thehost cell's endogenous plasmids.

Vectors useful for expression of nucleic acids in prokaryotes are wellknown in the art.

Isolated Polypeptides

According to another aspect of the present invention, isolatedpolypeptides (including muteins, allelic variants, fragments,derivatives, and analogs) encoded by the nucleic acid molecules of thepresent invention are provided. In one embodiment, the isolatedpolypeptide comprises the polypeptide sequence corresponding to SEQ IDNO:1, 2, 3, 7, 8, or 9. In an alternative embodiment of the presentinvention, the isolated polypeptide comprises a polypeptide sequence atleast 85% identical to SEQ ID NO:1, 2, 3, 7, 8, or 9. Preferably theisolated polypeptide of the present invention has at least 50%, 60, 70%,80%, 85%, 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%,98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or even higher identity to SEQ ID NO:1, 2, 3, 7, 8or 9.

According to other embodiments of the present invention, isolatedpolypeptides comprising a fragment of the above-described polypeptidesequences are provided. These fragments preferably include at least 20contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100 or even more contiguous amino acids.

The polypeptides of the present invention also include fusions betweenthe above-described polypeptide sequences and heterologous polypeptides.The heterologous sequences can, for example, include sequences designedto facilitate purification, e.g. histidine tags, and/or visualization ofrecombinantly-expressed proteins. Other non-limiting examples of proteinfusions include those that permit display of the encoded protein on thesurface of a phage or a cell, fusions to intrinsically fluorescentproteins, such as green fluorescent protein (GFP), and fusions to theIgG Fc region.

Host Cell Transformants

In another aspect of the present invention, host cells transformed withthe nucleic acid molecules or vectors of the present invention, anddescendants thereof, are provided. In some embodiments of the presentinvention, these cells carry the nucleic acid sequences of the presentinvention on vectors, which may but need not be freely replicatingvectors. In other embodiments of the present invention, the nucleicacids have been integrated into the genome of the host cells and/or intoan endogenous plasmid of the host cells.

In a preferred embodiment, the host cell comprises one or morerecombinant thioesterase-, acyl-CoA synthase-, wax synthase-, orTolC/AcrAB-encoding nucleic acids which express thioesterase-, acyl-CoAsynthase, wax synthase or TolC/AcrAB respectively in the host cell.

In an alternative embodiment, the host cells of the present inventioncan be mutated by recombination with a disruption, deletion or mutationof the isolated nucleic acid of the present invention so that theactivity of a native thioesterase, acyl-CoA synthase, wax synthase,and/or TolC/AcrAB protein in the host cell is reduced or eliminatedcompared to a host cell lacking the mutation.

Selected or Engineered Microorganisms for the Production of Fatty Acids,Esters, and Other Carbon-Based Products of Interest

Microorganism: Includes prokaryotic and eukaryotic microbial speciesfrom the Domains Archaea, Bacteria and Eucarya, the latter includingyeast and filamentous fungi, protozoa, algae, or higher Protista. Theterms “microbial cells” and “microbes” are used interchangeably with theterm microorganism.

A variety of host organisms can be transformed to produce a product ofinterest. Photoautotrophic organisms include eukaryotic plants andalgae, as well as prokaryotic cyanobacteria, green-sulfur bacteria,green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfurbacteria.

Extremophiles are also contemplated as suitable organisms. Suchorganisms withstand various environmental parameters such astemperature, radiation, pressure, gravity, vacuum, desiccation,salinity, pH, oxygen tension, and chemicals. They includehyperthermophiles, which grow at or above 80° C. such as Pyrolobusfumarii; thermophiles, which grow between 60-80° C. such asSynechococcus lividis; mesophiles, which grow between 15-60° C. andpsychrophiles, which grow at or below 15° C. such as Psychrobacter andsome insects. Radiation tolerant organisms include Deinococcusradiodurans. Pressure-tolerant organisms include piezophiles, whichtolerate pressure of 130 MPa. Weight-tolerant organisms includebarophiles. Hypergravity (e.g., >1 g) hypogravity (e.g., <1 g) tolerantorganisms are also contemplated. Vacuum tolerant organisms includetardigrades, insects, microbes and seeds. Dessicant tolerant andanhydrobiotic organisms include xerophiles such as Artemia salina;nematodes, microbes, fungi and lichens. Salt-tolerant organisms includehalophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina.pH-tolerant organisms include alkaliphiles such as Natronobacterium,Bacillus firmus OF4, Spirulina spp. (e.g., pH>9) and acidophiles such asCyanidium caldarium, Ferroplasma sp. (e.g., low pH). Anaerobes, whichcannot tolerate O₂ such as Methanococcus jannaschii; microaerophils,which tolerate some O₂ such as Clostridium and aerobes, which require O₂are also contemplated. Gas-tolerant organisms, which tolerate pure CO₂include Cyanidium caldarium and metal tolerant organisms includemetalotolerants such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn),Ralstonia sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael. Life onthe Edge: Amazing Creatures Thriving in Extreme Environments. New York:Plenum (1998) and Seckbach, J. “Search for Life in the Universe withTerrestrial Microbes Which Thrive Under Extreme Conditions.” InCristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds.,Astronomical and Biochemical Origins and the Search for Life in theUniverse, p. 511. Milan: Editrice Compositori (1997).

Plants include but are not limited to the following genera: Arabidopsis,Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus,Saccharum, Salix, Simmondsia and Zea.

Algae and cyanobacteria include but are not limited to the followinggenera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes,Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium,Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora,Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus,Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa,Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus,Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella,Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys,Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis,Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira,Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis,Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena,Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros,Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis,Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris,Characiopsis, Characium, Charales, Chilomonas, Chlainomonas,Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis,Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella,Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea,Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema,Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton,Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas,Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa,Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus,Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta,Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera,Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis,Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium,Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium,Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta,Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca,Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis,Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella,Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon,Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis,Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella,Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema,Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis,Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis,Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta,Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia,Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta,Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis,Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax,Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia,Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga,Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea,Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium,Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia,Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema,Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium,Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne,Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron,Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium,Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia,Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion,Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis,Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella,Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira,Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias,Microchaete, Microcoleus, Microcystis, Microglena, Micromonas,Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus,Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris,Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium,Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema,Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria,Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus,Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas,Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium,Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium,Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis,Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora,Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema,Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus,Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra,Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis,Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella,Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus,Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma,Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium,Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium,Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis,Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas,Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris,Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis,Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma,Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia,Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus,Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix,Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia,Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis,Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis,Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma,Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum,Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus,Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis,Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus,Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella,Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium,Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra,Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum,Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella,Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira,Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix,Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria,Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella,Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria,Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium,Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.

Additional cyanobacteria include members of the genus Chamaesiphon,Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis,Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus,Prochloron, Synechococcus, Synechocystis, Cyanocystis, Dermocarpella,Stanieria, Xenococcus, Chroococcidiopsis, Myxosarcina, Arthrospira,Borzia, Crinalium, Geitlerinemia, Leptolyngbya, Limnothrix, Lyngbya,Microcoleus, Oscillatoria, Planktothrix, Prochlorothrix, Pseudanabaena,Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaena,Anabaenopsis, Aphanizomenon, Cyanospira, Cylindrospermopsis,Cylindrospermum, Nodularia, Nostoc, Scylonema, Calothrix, Rivularia,Tolypothrix, Chlorogloeopsis, Fischerella, Geitieria, Iyengariella,Nostochopsis, Stigonema and Thermosynechococcus.

Green non-sulfur bacteria include but are not limited to the followinggenera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix,Herpetosiphon, Roseiflexus, and Thermomicrobium.

Green sulfur bacteria include but are not limited to the followinggenera:

Chlorobium, Clathrochloris, and Prosthecochloris.

Purple sulfur bacteria include but are not limited to the followinggenera: Allochromatium, Chromatium, Halochromatium, Isochromatium,Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa,Thiorhodococcus, and Thiocystis,

Purple non-sulfur bacteria include but are not limited to the followinggenera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium,Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum,Rodovibrio, and Roseospira.

Aerobic chemolithotrophic bacteria include but are not limited tonitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp.,Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp.,Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibriosp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligatelychemolithotrophic hydrogen bacteria such as Hydrogenobacter sp., ironand manganese-oxidizing and/or depositing bacteria such as Siderococcussp., and magnetotactic bacteria such as Aquaspirillum sp.

Archaeobacteria include but are not limited to methanogenicarchaeobacteria such as Methanobacterium sp., Methanobrevibacter sp.,Methanothermus sp., Methanococcus sp., Methanomicrobium sp.,Methanospirillum sp., Methanogenium sp., Methanosarcina sp.,Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanussp.; extremely thermophilic S-Metabolizers such as Thermoproteus sp.,Pyrodictium sp., Sulfolobus sp., Acidianus sp. and other microorganismssuch as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp.,Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp.,Mycobacteria sp., and oleaginous yeast.

Preferred organisms for the manufacture of esters according to themethods disclosed herein include: Arabidopsis thaliana, Panicumvirgatum, Miscanthus giganteus, and Zea mays (plants); Botryococcusbraunii, Chlamydomonas reinhardtii and Dunaliela salina (algae);Synechococcus sp PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp.PCC 6803, Thermosynechococcus elongatus BP-1 (cyanobacteria); Chlorobiumtepidum (green sulfur bacteria), Chloroflexus auranticus (greennon-sulfur bacteria); Chromatium tepidum and Chromatium vinosum (purplesulfur bacteria); Rhodospirillum rubrum, Rhodobacter capsulatus, andRhodopseudomonas palusris (purple non-sulfur bacteria).

Yet other suitable organisms include synthetic cells or cells producedby synthetic genomes as described in Venter et al. US Pat. Pub. No.2007/0264688, and cell-like systems or synthetic cells as described inGlass et al. US Pat. Pub. No. 2007/0269862.

Still, other suitable organisms include microorganisms that can beengineered to fix carbon dioxide, such as Escherichia coli, Acetobacteraceti, Bacillus subtilis, yeast and fungi such as Clostridiumljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pseudomonas fluorescens, or Zymomonas mobilis.

The capability to use carbon dioxide as the sole source of cell carbon(autotrophy) is found in almost all major groups of prokaryotes. The CO₂fixation pathways differ between groups, and there is no cleardistribution pattern of the four presently-known autotrophic pathways.See, e.g., Fuchs, G. 1989. Alternative pathways of autotrophic CO ₂fixation, p. 365-382, in H. G. Schlegel, and B. Bowien (ed.),Autotrophic bacteria. Springer-Verlag, Berlin, Germany. The reductivepentose phosphate cycle (Calvin-Bassham-Benson cycle) represents the CO₂fixation pathway in almost all aerobic autotrophic bacteria, forexample, the cyanobacteria.

For producing esters via the recombinant expression of thioesterase,acyl-CoA synthetase and/or wax synthase enzymes, an engineeredcyanobacteria, e.g., a Synechococcus or Thermosynechococcus species, isespecially preferred. Other preferred organisms include Synechocystis,Klebsiella oxytoca, Escherichia coli or Saccharomyces cerevisiae. Otherprokaryotic, archaea and eukaryotic host cells are also encompassedwithin the scope of the present invention. Engineered ester-producingorganisms expressing thioesterase, acyl-CoA synthetase and/or waxsynthase enzymes can be further engineered to express recombinantTolC/AcrAB to enhance the extracellular transport of esters.

Carbon-Based Products of Interest: Esters

In various embodiments of the invention, desired esters or a mixturethereof can be produced. For example, by including a particular alcoholor mixture of alcohols in the culture media, methyl esters, ethylesters, propyl esters, butyl esters, and esters of higher chain lengthalcohols (or mixtures thereof, depending on the substrate alcoholsavailable to the photosynthetic microbe) can be synthesized. The carbonchain lengths of the esters can vary from C₁₀ to C₂₀, e.g., usingethanol as a substate, diverse esters including, e.g., ethyl myristate,ethyl palmitate, ethyl oleate, and/or ethyl stearate and/or mixturesthereof can be produced by a single engineered photosyntheticmicroorganism of the invention. Accordingly, the invention providesmethods and compositions for the production of various chain lengths ofesters, each of which is suitable for use as a fuel or any otherchemical use.

In preferred aspects, the methods provide culturing host cells fordirect product secretion for easy recovery without the need to extractbiomass. These carbon-based products of interest are secreted directlyinto the medium. Since the invention enables production of variousdefined chain length of hydrocarbons and alcohols, the secreted productsare easily recovered or separated. The products of the invention,therefore, can be used directly or used with minimal processing.

Media and Culture Conditions

One skilled in the art will recognize that a variety of media andculture conditions can be used in conjunction with the methods andengineered cyanobacteria disclosed herein for the bioproduction of fattyacid esters (see, e.g., Rogers and Gallon, Biochemistry of the Algae andCyanobacteria, Clarendon Press Oxford (1988); Burlwe, Algal Culture:From Laboratory to Pilot Plant, Carnegie Institution of WashingtonPublication 600 Washington, D.C., (1961); and Round, F. E. The Biologyof the Algae. St Martin's Press, New York, 1965; Golden S S et al.(1987) Methods Enzymol 153:215-231; Golden and Sherman, J. Bacteriology158:36 (1984), each of which is incorporated herein by reference).Exemplary culture conditions and media are also described in, e.g.,WO/2010/068288, filed May 21, 2009, published Jun. 17, 2010, andincorporated by reference herein. Typical culture conditions for themethods of the present invention include the use of JB 2.1 culture mediaor A+ media. A recipe for one liter of JB 2.1 appears in Table A, below.

TABLE A JB 2.1 media (1L) Chemical mg/L added FW Molarity Units SourceNaCl 18000 58.44 308 mM Fisher KCl 600 74.55 8.05 mM Fisher NaNO₃ 400084.99 47.06 mM Sigma Aldrich MgSO₄—7H₂O 5000 246.47 20.29 mM SigmaAldrich KH₂PO₄ 200 136.09 1.47 mM Fisher CaCl₂ 266 110.99 2.40 mM SigmaNaEDTA_(tetra) 30 372.24 80.59 μM Fisher Ferric Citrate 14.1 244.9557.48 μM Acros Organics Tris 1000 121.14 8.25 mM Fisher Vitamin B₁₂0.004 1355.37 2.95E−03 μM Sigma Aldrich (Cyanocobalamin) H₃BO₃ 34 61.83554 μM Acros Organics MnCl₂—4H₂O 4.3 197.91 21.83 μM Sigma ZnCl 0.32136.28 2.31 μM Sigma MoO₃ 0.030 143.94 0.21 μM Sigma Aldrich CuSO₄—5H₂O0.0030 249.69 0.012 μM Sigma Aldrich CoCl₂—6H₂O 0.012 237.93 0.051 μMSigma

As described in more detail in the Examples, below, in certainembodiments one or more alcohols (e.g., methanol, ethanol, propanol,butanol, etc.) may be added during culturing to produce the desiredfatty acid ester(s) of interest (e.g., a fatty acid methyl ester, afatty acid ethyl ester, etc., and mixtures thereof). For organisms thatrequire or metabolize most efficiently in the presence of light andcarbon dioxide, either carbon dioxide or bicarbonate can be used duringculturing.

Fuel Compositions

In various embodiments, compositions produced by the methods of theinvention are used as fuels. Such fuels comply with ASTM standards, forinstance, standard specifications for diesel fuel oils D 975-09b, andJet A, Jet A-1 and Jet B as specified in ASTM Specification D. 1655-68.Fuel compositions may require blending of several products to produce auniform product. The blending process is relatively straightforward, butthe determination of the amount of each component to include in a blendis much more difficult. Fuel compositions may, therefore, includearomatic and/or branched hydrocarbons, for instance, 75% saturated and25% aromatic, wherein some of the saturated hydrocarbons are branchedand some are cyclic. Preferably, the methods of the invention produce anarray of hydrocarbons, such as C₁₃-C₁₇ or C₁₀-C₁₅ to alter cloud point.Furthermore, the compositions may comprise fuel additives, which areused to enhance the performance of a fuel or engine. For example, fueladditives can be used to alter the freezing/gelling point, cloud point,lubricity, viscosity, oxidative stability, ignition quality, octanelevel, and flash point. Fuels compositions may also comprise, amongothers, antioxidants, static dissipater, corrosion inhibitor, icinginhibitor, biocide, metal deactivator and thermal stability improver.

In addition to many environmental advantages of the invention such asCO₂ conversion and renewable source, other advantages of the fuelcompositions disclosed herein include low sulfur content, low emissions,being free or substantially free of alcohol and having high cetanenumber.

Carbon Fingerprinting

Biologically-produced carbon-based products, e.g., ethanol, fatty acids,alkanes, isoprenoids, represent a new commodity for fuels, such asalcohols, diesel and gasoline. Such biofuels have not been producedusing biomass but use CO2 as its carbon source. These new fuels may bedistinguishable from fuels derived form petrochemical carbon on thebasis of dual carbon-isotopic fingerprinting. Such products,derivatives, and mixtures thereof may be completely distinguished fromtheir petrochemical derived counterparts on the basis of ¹⁴C (fM) anddual carbon-isotopic fingerprinting, indicating new compositions ofmatter.

There are three naturally occurring isotopes of carbon: ¹²C, ¹³C, and¹⁴C. These isotopes occur in above-ground total carbon at fractions of0.989, 0.011, and 10⁻¹², respectively. The isotopes ¹²C and ¹³C arestable, while ¹⁴C decays naturally to ¹⁴N, a beta particle, and ananti-neutrino in a process with a half-life of 5730 years. The isotope¹⁴C originates in the atmosphere, due primarily to neutron bombardmentof ¹⁴N caused ultimately by cosmic radiation. Because of its relativelyshort half-life (in geologic terms), ¹⁴C occurs at extremely low levelsin fossil carbon. Over the course of 1 million years without exposure tothe atmosphere, just 1 part in 10⁵⁰ will remain ¹⁴C.

The ¹³C:¹²C ratio varies slightly but measurably among natural carbonsources. Generally these differences are expressed as deviations fromthe ¹³C:¹²C ratio in a standard material. The international standard forcarbon is Pee Dee Belemnite, a form of limestone found in SouthCarolina, with a ¹³C fraction of 0.0112372. For a carbon source α, thedeviation of the ¹³C:¹²C ratio from that of Pee Dee Belemnite isexpressed as: δ_(a)=(R_(a)/R_(s))−1, where R_(a)=¹³C:¹²C ratio in thenatural source, and R_(s)=¹³C:¹²C ratio in Pee Dee Belemnite, thestandard. For convenience, δ_(a) is expressed in parts per thousand, or

. A negative value of δ_(a) shows a bias toward ¹²C over ¹³C as comparedto Pee Dee Belemnite. Table 1 shows δ_(a) and ¹⁴C fraction for severalnatural sources of carbon.

TABLE 1 13C:12C variations in natural carbon sources Source −δ_(a) (‰)References Underground coal 32.5 Farquhar et al. (1989) Plant Mol.Biol., 40: 503-37 Fossil fuels 26 Farquhar et al. (1989) Plant Mol.Biol., 40: 503-37 Ocean DIC*   0-1.5 Goericke et al. (1994) Chapter 9 inStable Isotopes in Ecology and Environmental Science, by K. Lajtha andR. H. Michener, Blackwell Publishing; Ivlev (2010) Separation Sci.Technol. 36: 1819-1914 Atmospheric 6-8 Ivlev (2010) Separation Sci.Technol. 36: CO2 1819-1914; Farquhar et al. (1989) Plant Mol. Biol., 40:503-37 Freshwater DIC*  6-14 Dettman et al. (1999) Geochim. Cosmochim.Acta 63: 1049-1057 Pee Dee 0 Ivlev (2010) Separation Sci. Technol. 36:Belemnite 1819-1914 *DIC = dissolved inorganic carbon

Biological processes often discriminate among carbon isotopes. Thenatural abundance of ¹⁴C is very small, and hence discrimination for oragainst ¹⁴C is difficult to measure. Biological discrimination between¹³C and ¹²C, however, is well-documented. For a biological product p, wecan define similar quantities to those above: δ_(p)═(R_(p)/R_(s))−1,where R_(p)=¹³C:¹²C ratio in the biological product, and R_(s)=¹³C:¹²Cratio in Pee Dee Belemnite, the standard. Table 2 shows measureddeviations in the ¹³C:¹²C ratio for some biological products.

TABLE 2 ¹³C:¹²C variations in selected biological products Product−δ_(p) (‰) −D (‰)* References Plant sugar/starch from 18-28 10-20 Ivlev(2010) Separation Sci. atmospheric CO₂ Technol. 36: 1819-1914Cyanobacterial biomass from 18-31 16.5-31 Goericke et al. (1994) marineDIC Chapter 9 in Stable Isotopes in Ecology and Environmental Science,by K. Lajtha and R. H. Michener, Blackwell Publishing; Sakata et al.(1997) Geochim. Cosmochim. Acta, 61: 5379-89 Cyanobacterial lipid frommarine 39-40 37.5-40 Sakata et al. (1997) DIC Geochim. Cosmochim. Acta,61: 5379-89 Algal lipid from marine DIC 17-28 15.5-28 Goericke et al.(1994) Chapter 9 in Stable Isotopes in Ecology and EnvironmentalScience, by K. Lajtha and R. H. Michener, Blackwell Publishing; Abelseonet al. (1961) Proc. Natl. Acad. Sci., 47: 623-32 Algal biomass fromfreshwater 17-36   3-30 Marty et al. (2008) Limnol. DIC Oceanogr.:Methods 6: 51-63 E. coli lipid from plant sugar 15-27 near 0 Monson etal. (1980) J. Biol. Chem., 255: 11435-41 Cyanobacterial lipid fromfossil 63.5-66   37.5-40 — carbon Cyanobacterial biomass from 42.5-57  16.5-31 — fossil carbon *D = discrimination by a biological process inits utilization of ¹²C vs. ¹³C (see text)

Table 2 introduces a new quantity, D. This is the discrimination by abiological process in its utilization of ¹²C vs. ¹³C. We define D asfollows: D=(R_(p)/R_(a))−1. This quantity is very similar to δ_(a) andδ_(p), except we now compare the biological product directly to thecarbon source rather than to a standard. Using D, we can combine thebias effects of a carbon source and a biological process to obtain thebias of the biological product as compared to the standard. Solving forδ_(p), we obtain: δ_(p)=(D)(δ_(a))+D+δ_(a), and, because (D)(δ_(a)) isgenerally very small compared to the other terms, δ_(p)≈δ_(a)+D.

For a biological product having a production process with a known D, wemay therefore estimate δ_(p) by summing δ_(a) and D. We assume that Doperates irrespective of the carbon source. This has been done in Table1 for cyanobacterial lipid and biomass produced from fossil carbon. Asshown in the Table 1 and Table 2, above, cyanobacterial products madefrom fossil carbon (in the form of, for example, flue gas or otheremissions) will have a higher δ_(p) than those of comparable biologicalproducts made from other sources, distinguishing them on the basis ofcomposition of matter from these other biological products. In addition,any product derived solely from fossil carbon will have a negligiblefraction of ¹⁴C, while products made from above-ground carbon will havea ¹⁴C fraction of approximately 10⁻¹².

Accordingly, in certain aspects, the invention provides variouscarbon-based products of interest characterized as −δ_(p)(

) of about 63.5 to about 66 and −D(

) of about 37.5 to about 40.

The following examples are for illustrative purposes and are notintended to limit the scope of the present invention.

EXAMPLE 1 Recombinant Genes for the Biosynthesis of Biodiesel andBiodiesel-Like Compounds

In one embodiment of the invention, a cyanobacterium strain istransformed or engineered to express one or more enzymes selected fromthe following list: a wax synthase (EC: 2.3.175), a thioesterase (EC:3.1.2.-, 3.1.2.14), and an acyl-CoA synthase (EC:: 6.2.1.3). Forexample, a typical embodiment utilizes a thioesterase gene from E. coli(tesA; SEQ ID NO:1), an acyl-CoA synthetase gene from E. coli (fadD; SEQID NO:2), and a wax synthase gene from A. baylyi (wax; SEQ ID NO:3).Thioesterase generates fatty acid from acyl-ACP. Acyl-CoA synthetase(also referred to as acyl-CoA ligase) generates fatty acyl-CoA fromfatty acid. Wax synthase (EC 2.3.1.75) generates fatty acid esters usingacyl-CoA and acyl alcohol as substrates (e.g., methanol, ethanol,butanol, etc).

Additional thioesterase, acyl-CoA synthetase and wax synthases genesthat can be recombinantly expressed in cyanobacteria are set forth inTable 3A, Table 3B, and Table 3C, respectively.

TABLE 3A Exemplary Thioesterases* Genbank: GenBank: gene proteinaccession accession Source Enzyme number number E. coli C-18:1thioesterase NC_000913 NP_415027 Cuphea C-8:0 to C-10:0 thioesteraseU39834.1 AAC49269 hookeriana Umbellularia C-12:0 thioesterase M94159.1Q41635 california Cinnamonum C-14:0 thioesterase U17076.1 Q39473camphorum Arabidopsis C-18:1 thioesterase 822102 NP_189147.1 thaliana*where leader sequences are present in the native protein, as in thecase of E. coli tesA, the leader sequences are typically removed beforethe activity is recombinantly expressed

TABLE 3B Exemplary Acyl-CoA Synthetases Genbank: GenBank: protein geneaccession accession Source Gene name number number E. coli Acyl-CoANC_000913 NP_416319.1 synthetase Geobacillus Acyl-CoA CP000557.1ABO66726.1 thermodenitrificans synthetase NG80-2

TABLE 3C Exemplary Wax Synthases Genbank: GenBank: protein Gene or geneaccession accession Source protein name number number Acinetobacterbaylyi wxs AF529086.1 AAO17391.1 Mycobacterium acyltransferase,NP_218257.1 tuberculosis H37Rv WS/DGAT/ MGAT Saccharomyces Eeb1NP_015230 cerevisiae Saccharomyces YMR210w NP_013937 cerevisiae Rattusnorvegicus FAEE synthase P16303 (rat) Fundibacter jadensis wst9 DSM12178 Acinetobacter sp. Wshn H01-N H. sapiens mWS Fragaria xananassaSAAT Malus xdomestica mpAAT Simmondsia chinensis JjWs Q9XGY6 Musmusculus mWS Q6E1M8

The engineered cyanobacterium expressing one or more of thethioesterase, acyl-CoA synthetase, and wax synthase genes set forthabove is grown in suitable media, under appropriate conditions (e.g.,temperature, shaking, light, etc.). After a certain optical density isreached, the cells are separated from the spent medium bycentrifugation. The cell pellet is re-suspended and the cell suspensionand the spent medium are then extracted with a suitable solvent, e.g.,ethyl acetate. The resulting ethyl acetate phases from the cellsuspension and the supernatant are subjected to GC-MS analysis. Thefatty acid esters in the ethyl acetate phases can be quantified, e.g.,using commercial palmitic acid ethyl ester as a reference standard.

Fatty acid esters can be made according to this method by adding analcohol (e.g., methanol, propanol, isopropanol, butanol, etc.) to thefermentation media, whereby fatty acid esters of the added alcohols areproduced by the engineered cyanobacterium. Alternatively, one or morealcohols can be synthesized by the engineered cyanobacterium, nativelyor recombinantly, and used as substrates for fatty acid ester synthesisby a recombinantly expressed wax synthase. As detailed in the Examplesbelow, the engineered cyanobacterium can also be modified torecombinantly express a TolC/AcrAB transporter to facilitate secretionof the fatty acid esters into the culture medium.

EXAMPLE 2 Synthesis of Ethyl and Methyl Fatty Acid Esters by anEngineered Cyanobacterium

Genes and Plasmids: The pJB5 base vector was designed as an emptyexpression vector for recombination into Synechococcus sp. PCC 7002. Tworegions of homology, the Upstream Homology Region (UHR) and theDownstream Homology Region (DHR), are designed to flank the construct ofinterest. These 500 bp regions of homology correspond to positions3301-3800 and 3801-4300 (Genbank Accession NC_(—)005025) for UHR and DHRrespectively. The aadA promoter, gene sequence, and terminator weredesigned to confer spectinomycin and streptomycin resistance to theintegrated construct. For expression, pJB5 was designed with the aphIIkanamycin resistance cassette promoter and ribosome binding site (RBS).Downstream of this promoter and RBS, the restriction endonucleaserecognition site for NdeI, EcoRI, SpeI and PacI were inserted. Followingthe EcoRI site, the natural terminator from the alcohol dehydrogenasegene from Zymomonas mobilis (adhII) terminator was included. ConvenientXbaI restriction sites flank the UHR and the DHR allowing cleavage ofthe DNA intended for recombination from the rest of the vector.

The E. coli thioesterase tesA gene with the leader sequence removed (SEQID NO:4; Genbank #NC_(—)000913; Chot and Cronan, 1993), the E. coliacyl-CoA synthetase fadD (SEQ ID NO:5; Genbank #NC_(—)000913; Kameda andNunn, 1981) and the wax synthase gene (wax) from Acinetobacter baylyistrain ADPI (SEQ ID NO:6; Genbank #AF529086.1; Stöveken et al. 2005)were purchased from DNA 2.0, following codon optimization, checking forsecondary structure effects, and removal of any unwanted restrictionsites (NdeI, XhoI, BamHI, NgoMIV, NcoI, SacI, BsrGI, AvrII, BmtI, MluI,EcoRI, SbfI, NotI, SpeI, XbaI, PacI, AscI, FseI). These genes werereceived on a pJ201 vector and assembled into a three-gene operon(tesA-fadD-wax, SEQ ID NO: 10) with flanking NdeI-EcoRI sites on therecombination vector pJB5 under the control of the PaphII kanamycinresistance cassette promoter. A second plasmid (pJB532; SEQ ID NO:11)was constructed which is identical to pJB494 except the PaphII promoterwas replaced with SEQ ID NO:12, a Ptrc promoter and a lacIq repressor.As a control, a third plasmid (pJB413) was prepared with only tesA underthe control of the PaphII promoter. These plasmid constructs were namedpJB494, pJB532, and pJB413, respectively.

Strain Construction: The constructs described above were integrated ontothe plasmid pAQ1 in Synechococcus sp. PCC 7002 according to thefollowing protocol. Synechococcus 7002 was grown for 48 h from coloniesin an incubated shaker flask at 37° C. at 2% CO₂ to an OD₇₃₀ of 1 in A⁺medium described in Frigaard et al., Methods Mol. Biol., 274:325-340(2004). 450 μL of culture was added to a epi-tube with 50 μL of 5 μg ofplasmid DNA digested with XbaI ((New England Biolabs; Ipswitch, Mass.))that was not purified following restriction digest. Cells were incubatedin the dark for four hours at 37° C. The entire volume of cells wasplated on A⁺ medium plates with 1.5% agarose and grown at 37° C. in alighted incubator (40-60 μE/m2/s PAR, measured with a LI-250A lightmeter (LI-COR)) for about 24 hours. 25 μg/mL of spectinomycin wasunderlayed on the plates. Resistant colonies were visible in 7-10 daysafter further incubation, and recombinant strains were confirmed by PCRusing internal and external primers to check insertion and confirmlocation of the genes on pAQ1 in the strains (Table 4).

TABLE 4 Joule Culture Collection (JCC) numbers of Synechococcus sp. PCC7002 recombinant strains with gene insertions on the native plasmid pAQ1JCC # Promoter Genes Marker JCC879 PaphII — aadA JCC750 PaphII tesA aadAJCC723 PaphII tesA-fadD-wax aadA JCC803 lacIq Ptrc tesA-fadD-wax aadA

Ethyl Ester Production culturing conditions: One colony of each of thefour strains listed in Table 4 was inoculated into 10 ml of A+ mediacontaining 50 μg/ml spectinomycin and 1% ethanol (v/v). These cultureswere incubated for about 4 days in a bubble tube at 37° C. sparged atapproximately 1-2 bubbles of 1% CO₂/air every 2 seconds in light (40-50μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)). The cultureswere then diluted so that the following day they would have OD₇₃₀ of2-6. The cells were washed with 2×10 ml JB 2.1/spec200, and inoculatedinto duplicate 28 ml cultures in JB 2.1/spec200+1% ethanol (v/v) mediato an OD₇₃₀=0.07. IPTG was added to the JCC803 cultures to a finalconcentration of 0.5 mM. These cultures were incubated in a shakingincubator at 150 rpm at 37° C. under 2% CO₂/air and continuous light(70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR)) forten days. Water loss through evaporation was replaced with the additionof sterile Milli-Q water. 0.5% (v/v) ethanol was added to the culturesto replace loss due to evaporation every 48 hours. At 68 and 236 hours,5 ml and 3 ml of culture were removed from each flask for ethyl esteranalysis, respectively. The OD₇₃₀ values reached by the cultures aregiven in Table 5.

TABLE 5 OD₇₃₀s reached by recombinant Synechococcus sp. PCC 7002 strainsat timepoints 68 and 236 h JCC879 JCC879 JCC750 JCC750 JCC723 JCC723JCC803 JCC803 Time point #1 #2 #1 #2 #1 #2 #1 #2 68 h 3.6 4.0 4.6 5.06.6 6.0 5.4 5.8 236 h 21.2 18.5 19.4 20.9 22.2 21.4 17.2 17.7

The culture aliquots were pelleted using a Sorvall RC6 Plus superspeedcentrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor (5000 rpm for10 min). The spent media supernatant was removed and the cells wereresuspended in 1 ml of Milli-Q water. The cells were pelleted againusing a benchtop centrifuge, the supernatant discarded and the cellpellet was stored at −80° C. until analyzed for the presence of ethylesters.

Detection and quantification of ethyl esters in strains: Cell pelletswere thawed and 1 ml aliquots of acetone (Acros Organics 326570010)containing 100 mg/L butylated hydroxytoluene (Sigma-Aldrich B1378) and50 mg/L ethyl valerate (Fluka 30784) were added. The cell pellets weremixed with the acetone using a Pasteur pipettes and vortexed twice for10 seconds (total extraction time of 1-2 min). The suspensions werecentrifuged for 5 min to pellet debris, and the supernatants wereremoved with Pasteur pipettes and subjected to analysis with a gaschromatograph using flame ionization detection (GC/FID).

An Agilent 7890A GC/FID equipped with a 7683 series autosampler was usedto detect the ethyl esters. One μL of each sample was injected into theGC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time:0.2 min, purge flow: 15 mL/min) and an inlet temperature of 280° C. Thecolumn was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gaswas helium at a flow of 1.0 mL/min. The GC oven temperature program was50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes.The GC/MS interface was 290° C., and the MS range monitored was 25 to600 amu. Ethyl myristate [C14:0; retention time (rt): 17.8 min], ethylpalmitate (C16:0; rt: 19.8 min) and ethyl stearate (C18:0; rt: 21.6 min)were identified based on comparison to a standard mix of C₄-C₂₄ evencarbon saturated fatty acid ethyl esters (Supelco 49454-U). Ethyl oleate(C18:1; rt: 21.4 min) was identified by comparison with an ethyl oleatestandard (Sigma Aldrich 268011). These identifications were confirmed byGC/MS (see following Methyl Ester Production description for details).Calibration curves were constructed for these ethyl esters using thecommercially available standards, and the concentrations of ethyl esterspresent in the extracts were determined and normalized to theconcentration of ethyl valerate (internal standard).

Four different ethyl esters were found in the extracts of JCC723 andJCC803 (Table 6 and Table 7). In general, JCC803 produced 2-10× theamount of each ethyl ester than JCC723, but ethyl myristate (C14:0) wasonly produced in low quantities of 1 mg/L or less for all thesecultures. Both JCC723 and JCC803 produced ethyl esters with the relativeamounts C16:0>C18:0>C18:1 (cis-9)>C14:0. No ethyl esters were found inthe extracts of JCC879 or JCC750, indicating that the strain cannot makeethyl esters naturally and that expression of only the tesA gene is notsufficient to confer production of ethyl esters.

TABLE 6 Amounts of respective ethyl esters found in the cell pelletextracts of JCC723 given as mg/L of culture C18:1 C14:0 C16:0 (cis-9)C18:0 Sample myristate palmitate oleate stearate % Yield* JCC723 #1 68 h0.08 0.34 0.22 0.21 0.04 JCC723 #2 68 h 0.12 1.0 0.43 0.40 0.1 JCC803 #168 h 0.45 6.6 1.4 0.74 0.6 JCC803 #2 68 h 0.63 8.6 2.0 0.94 0.7 JCC723#1 236 h 1.04 15.3 2.1 4.5 0.3 JCC723 #2 236 h 0.59 9.0 1.3 3.7 0.2JCC803 #1 236 h 0.28 35.3 13.4 19.2 1.3 JCC803 #2 236 h 0.49 49.4 14.921.2 1.6 *Yield (%) = ((sum of EEs)/dry cell weight) * 100

TABLE 7 % of total ethyl esters by mass C14:0 C16:0 C18:1 Samplemyristate palmitate oleate C18:0 stearate JCC723 #1  68 h 9.4 40.0 25.924.7 JCC723 #2  68 h 6.2 51.3 22.1 20.5 JCC803 #1  68 h 4.9 71.8 15.28.1 JCC803 #2  68 h 5.2 70.7 16.4 7.7 JCC723 #1 236 h 4.5 66.7 9.2 19.6JCC723 #2 236 h 4.0 61.7 8.9 25.4 JCC803 #1 236 h 0.4 51.8 19.7 28.2JCC803 #2 236 h 0.6 57.4 17.3 24.7

Methyl Ester Production Culturing conditions: One colony of JCC803(Table 1) was inoculated into 10 mL of A+ media containing 50 μg/mlspectinomycin and 1% ethanol (v/v). This culture was incubated for 3days in a bubble tube at 37° C. sparged at approximately 1-2 bubbles of1% CO₂/air every 2 seconds in light (40-50 μE/m2/s PAR, measured with aLI-250A light meter (LI-COR)). The culture was innoculated into twoflasks to a final volume of 20.5 ml and OD₇₃₀=0.08 in A+ mediacontaining 200 μg/ml spectinomycin and 0.5 mM IPTG with either 0.5%methanol or 0.5% ethanol (v/v). These cultures were incubated in ashaking incubator at 150 rpm at 37° C. under 2% CO₂/air and continuouslight (70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR))for three days. Water loss through evaporation was replaced with theaddition of sterile Milli-Q water. Samples of 5 ml of these cultures(OD₇₃₀=5-6) were analyzed for the presence of ethyl or methyl esters.

Detection of methyl esters and comparison with ethyl ester production inthe same strain: Cell pellets were thawed and 1 ml aliquots of acetone(Acros Organics 326570010) containing 100 mg/L butylated hydroxytoluene(Sigma-Aldrich B 1378) and 50 mg/L ethyl valerate (Fluka 30784) wereadded. The cell pellets were mixed with the acetone using a Pasteurpipette and vortexed twice for 10 seconds (total extraction time of 1-2min). The suspensions were centrifuged for 5 min to pellet debris, andthe supernatants were removed with Pasteur pipettes and subjected toanalysis with a gas chromatograph using mass spectral detection (GC/MS).

An Agilent 7890A GC/5975C EI-MS equipped with a 7683 series autosamplerwas used to measure the ethyl esters. One μL of each sample was injectedinto the GC inlet using pulsed splitless injection (pressure: 20 psi,pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and aninlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 mL/min. TheGC oven temperature program was 50° C., hold one minute; 10°/minincrease to 280° C.; hold ten minutes. The GC/MS interface was 290° C.,and the MS range monitored was 25 to 600 amu. Compounds indicated bypeaks present in total ion chromatograms were identified by matchingexperimentally determined mass spectra associated with the peaks withmass spectral matches found by searching in a NIST 08 MS database.

The culture of JCC803 incubated with ethanol contained ethyl palmitate[C16:0; retention time (rt): 18.5 min], ethyl heptadecanoate (C17rt:19.4 min), ethyl oleate (C18:1; rt: 20.1 min) and ethyl stearate (C18:0;rt: 20.3 min) (FIG. 1). The relative amounts produced wereC16:0>C18:0>C18:1>C17:0. The production of low levels of C17:0 and theabsence of measured levels of C14:0/myristate in this experiment islikely a result of the use of A+ medium (JB 2.1 was used to generate thedate in Table 7, above).

No ethyl esters were detected in the strain incubated with methanol.Instead, methyl palmitate (C16:0; retention time (“rt”): 17.8 min),methyl heptadecanoate (C17:0; rt: 18.8 min) and methyl stearate (C18:0)were found (FIG. 1; methyl palmitate: 0.1 mg/L; methyl heptadecanoate:0.062 mg/L; methyl stearate: 0.058 mg/L; total FAMEs: 0.22 mg/L; % ofDCW: 0.01).

The data presented herein shows that JCC803 and other cyanobacterialstrains engineered with tesA-fadD-wax genes can utilize methanol,ethanol, butanol, and other alcohols, including exogenously addedalcohols, to produce a variety of fatty acid esters. In certainembodiments, multiple types of exogenous or endogenous alcohols (e.g.,methanol and ethanol; butanol or ethanol; methanol and butanol; etc.)could be added to the culture medium and utilized as substrates.

EXAMPLE 3 Production of Fatty-Acid Esters Through HeterologousExpression of an Acyl-CoA Synthetase and a Wax Synthase

In order to compare the yields of fatty-acid esters produced byrecombinant strains expressing tesA-fadD or fadD-wax (i.e., two of thethree genes in the tesA-fadD-wax synthetic operon), fadD-wax andtesA-fadD and were assembled as two-gene operons and inserted into pJB5to yield pJB634 and pJB578, respectively. These recombination plasmidswere transformed into Synechococcus sp. PCC 7002 as described in Example1, above to generate the strains listed in Table 8. Table 8 also listsJCC723, described above.

TABLE 8 Joule Culture Collection (JCC) numbers of the Synechococcus sp.PCC 7002 recombinant strains with gene insertions on the native plasmidpAQ1. Promoter-operon % DCW Strain # Promoter Genes sequences MarkerOD₇₃₀ FAEE JCC723 PaphII tesA-fadD-wax SEQ ID NO: 10 aadA 15.35 0.20JCC1215 PaphII fadD-wax SEQ ID NO: 13 aadA 10.10 0.04 JCC1216 PaphIItesA-fadD SEQ ID NO: 14 aadA 10.00 0.00

One 30-ml culture of each strain listed in Table 1 was prepared in JB2.1 medium containing 200 mg/L spectinomycin and 1% ethanol (vol/vol) atan OD₇₃₀=0.1 in 125 ml flasks equipped with foam plugs (inocula werefrom five ml A+ cultures containing 200 mg/L spectinomyin started fromcolonies incubated for 3 days in a Multitron II Infors shakingphotoincubator under continuous light of ˜100 μE m⁻²s⁻¹photosynthetically active radiation (PAR) at 37° C. at 150 rpm in 2%CO₂-enriched air). The cultures were incubated for seven days in theInfors incubators under continuous light of ˜100 μE m⁻²s⁻¹photosynthetically active radiation (PAR) at 37° C. at 150 rpm in 2%CO₂-enriched air. Fifty percent of the starting volume of ethanol wasadded approximately at day 5 based on experimentally determinedstripping rates of ethanol under these conditions. Water loss wascompensated by adding back milli-Q water (based on weight loss offlasks). Optical density measurements at 730 nm (OD₇₃₀) were taken(Table 8), and esters were extracted from cell pellets using the acetoneprocedure detailed in Example 2, above. Ethyl arachidate (Sigma A9010)at 100 mg/L was used as an internal standard instead of ethyl valerate.The dry cell weights (DCWs) were estimated based on the OD measurementusing an experimentally determined average of 300 mg L⁻¹ OD₇₃₀ ⁻¹.

The acetone extracts were analyzed by GC/FID (for instrument conditions,see Example 2). In order to quantify the various esters, responsefactors (RF) were estimated from RFs measured for authentic ethyl esterstandards and these RFs were used to determine the titres in the acetoneextracts. The % DCW of the fatty-acid esters and the sum of the estersas % DCW is given in Table 8. Expression of fadD-wax was sufficient toallow production of fatty-acid ethyl esters (FAEEs), while expression oftesA-fadD did not result in any FAEEs (FIG. 2). The overall yield waslower than JCC723, indicating that the co-expression of tesA isbeneficial for increasing yields of FAEEs in this strain.

EXAMPLE 4 Production of Longer-Chain Fatty-Acid Esters by Addition ofRespective Alcohols to tesA-fadD-Wax Cultures

Seven 30-ml cultures of JCC803 (prepared from a single JCC803 culturethat was diluted into 250 ml of JB 2.1 media containing 200 mg/Lspectinomycin at an OD₇₃₀=0.1) in 125-ml flasks were used to evaluatethe ability of JCC803 to esterify different alcohols with fatty acids.Seven different alcohols were added at concentrations previouslydetermined to allow growth of JCC803 (Table 9). The cultures wereincubated for seven days in a Multitron II Infors shaking photoincubatorunder continuous light of ˜100 μE m⁻²s⁻¹ photosynthetically activeradiation (PAR) at 37° C. at 150 rpm in 2% CO₂-enriched air. Water losswas compensated by adding back milli-Q water (based on weight loss offlasks). Optical density measurements at 730 nm (OD₇₃₀) were taken(Table 3), and esters were extracted from cell pellets using the acetoneprocedure detailed in Example 2, above. Ethyl arachidate (Sigma A9010)at 100 mg/L was used as an internal standard instead of ethyl valerate.The dry cell weights (DCWs) were also determined for each culture sothat the % DCW of the esters could be reported.

TABLE 9 Concentration Final Alcohol Catalog # % (vol/vol) OD₇₃₀ Propanol256404 (Sigma) 0.25 12.6 Isopropanol BP2632 (Fisher) 0.25 12.6 Butanol34867 (Sigma) 0.1 12.5 Hexanol H13303 (Sigma) 0.01 8.6 Cyclohexanol105899 (Sigma) 0.01 13.6 Isoamyl alcohol A393 (Fisher) 0.05 13.6 Ethanol2716 (Decon Labs Inc.) 1.0 14.0

The acetone extracts were analyzed by GC/MS and GC/FID, as describedabove. The compounds indicated by peaks present in the total ionchromatograms were identified by matching the mass spectra associatedwith the peaks with mass spectral matches found by searching the NIST 08MS database or by interpretation of the mass spectra when a respectivemass spectrum of an authentic standard was not available in thedatabase. In all cases, the corresponding alcohol esters of fatty acidswere produced by JCC803 (FIG. 3). Six fatty-acid esters were detectedand quantified in the cell pellet extracts: myristate (C14:0),palmitoleate (C16:1Δ9), palmitate (C16:0), margarate (C17:0), oleate(C18:1Δ9) and stearate (C18:0). Magnified chromatograms for JCC803incubated with ethanol and butanol are shown in FIG. 4 and FIG. 5,respectively, so that the lower-yielding palmitoleate and margarateesters could be indicated on the chromatograms. In order to quantify thevarious esters, response factors (RF) were estimated from RFs measuredfor authentic ethyl ester and these RFs were used to determine thetitres in the acetone extracts. The % DCW of the different esters andthe sum of the esters as % DCW is given in Table 10. The % of theindividual esters by weight and the total ester yield in mg/L is givenin Table 11.

In general, the provision of longer-chain alcohols increased the yieldsof fatty-acid esters. The addition of butanol resulted in the highestyields of fatty-acid esters. Because butanol can be madebiosynthetically (Nielsen et al. 2009, and references therein),exogenous butanol biosynthetic pathways could be expressed by oneskilled in the art to generate a photosynthetic strain which can producebutyl esters without the addition of butanol. The use of butanol andbutanol-producing pathways in other microbes containing thetesA-fadD-wax pathway would also be expected to increase yields offatty-acid esters.

TABLE 10 The yield of the fatty acid-esters individually and total as %dry cell weight Total Myristate Palmitoleate Palmitate Margarate OleateStearate Ester Ethyl 0.05 0.02 0.94 0.01 0.11 0.15 1.3 Propyl 0.26 0.063.22 0.03 0.21 0.48 4.3 Isopropyl 0.20 0.04 2.42 0.02 0.08 0.42 3.2Butyl 0.59 0.06 3.67 0.03 0.19 0.56 5.1 Hexyl 0.11 0.04 1.33 0.02 0.170.19 1.8 Cyclohexyl 0.09 0.03 1.88 0.01 0.09 0.31 2.4 Isoamyl 0.31 0.052.84 0.02 0.15 0.46 3.8

TABLE 11 The % of the individual esters by weight and total ester yieldin mg/L. Total Myristate Palmitoleate Palmitate Margarate OleateStearate Ester Ethyl 4.2 1.2 73.4 0.7 8.6 12.0 77.6 Propyl 6.0 1.3 76.00.7 4.9 11.1 251.7 Isopropyl 6.2 1.2 76.4 0.8 2.4 13.0 188.5 Butyl 11.41.1 72.6 0.5 3.7 10.8 308.9 Hexyl 6.0 2.1 71.9 1.1 8.9 10.0 65.3Cyclohexyl 3.6 1.1 78.5 0.6 3.6 12.7 139.6 Isoamyl 8.1 1.2 74.6 0.5 3.911.8 226.8

EXAMPLE 5 Reproducibility of Butanol Yields in tesA-fadD-wax Cultures

Six 30-ml cultures of JCC803 (prepared from a single JCC803 culture thatwas diluted into 200 ml of JB 2.1 media/spec200 at an OD₇₃₀=0.1) in 125ml flasks were used to evaluate the ability of JCC803 cultures toproduce butyl esters when containing different concentrations ofbutanol. Six different concentrations were tested (Table 12). Thecultures were incubated for 21 days in a Multitron II Infors shakingphotoincubator under continuous light at ˜100 μE m⁻²s⁻¹ PAR at 37° C. at150 rpm in 2% CO₂-enriched air. Fifty percent of the starting volume ofbutanol was added approximately every 3.5 days based on experimentallydetermined stripping rates of butanol under these conditions. Water losswas compensated by adding back milli-Q water (based on weight loss offlasks). OD₇₃₀s were taken and esters were extracted from cell pelletsusing the acetone procedure detailed above. 100 mg/L ethyl arachidate(Sigma A9010) was used as an internal standard instead of ethylvalerate. The dry cell weights (DCWs) were also determined for eachculture so that the % DCW of the esters could be reported.

An Agilent 7890A GC/FID equipped with a 7683 series autosampler was usedto quantify the butyl esters. One microliter of each sample was injectedinto the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min,purge time: 0.2 min, purge flow: 15 mL/min), which was at a temperatureof 280° C. The column was an HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm), andthe carrier gas was helium at a flow of 1.0 mL/min. The GC oventemperature program was: 50° C., hold one minute; 10°/min increase to280° C.; hold ten minutes. Butyl myristate, butyl palmitate, butylmargarate, butyl oleate and butyl stearate were quantified bydetermining appropriate response factors for the number of carbonspresent in the butyl esters from commercially available fatty-acid ethylesters (“FAEEs”) and fatty acid butyl esters (“FABEs”). The calibrationcurves were prepared for ethyl laurate (Sigma 61630), ethyl myristate(Sigma E39600), ethyl palmitate (Sigma P9009), ethyl oleate (Sigma268011), ethyl stearate (Fluka 85690), butyl laurate (Sigma W220604) andbutyl stearate (Sigma S5001). The concentrations of the butyl esterspresent in the extracts were determined and normalized to theconcentration of ethyl arachidate (internal standard).

The yields of the JCC803 cultures as given by the % DCW of the fattyacid butyl esters is given in Table 12. The highest yield of 14.7%resulted from the culture incubated with 0.05% butanol (vol/vol)although the 0.075% butanol-containing culture was approximately thesame.

TABLE 12 Yield of total FABES as % DCW for the JCC803 culturescontaining different concentrations of butanol and final OD₇₃₀ of thecultures. Concentration of butanol % (vol/vol) OD₇₃₀ % DCW 0.2 10.611.75 0.1 9.0 12.43 0.075 12.8 14.53 0.05 12.0 14.71 0.025 13.4 10.430.01 16.0 6.12

EXAMPLE 6 Secretion of Esters Produced by an Engineered Cyanobacterium

Plasmids. Escherichia coli exports alkanes and other hydrophobicmolecules out of the cell via the TolC-AcrAB transporter complex(Tsukagoshi and Aono, 2000; Chollet et al. 2004). PCR primer sets weredesigned to amplify tolC (Genbank #NC_(—)000913.2, locus b3035) andacrA-acrB as an operon (Genbank #NC_(—)000913.2, loci b0463, b0462) fromE. coli MG1655 (ATCC #700926). The tolC and acrAB genes were amplifiedfrom MG1655 genomic DNA using the Phusion High-Fidelity PCR kit F-553from New England BioLabs (Ipswich, Mass.) following the manufacturer'sinstructions. Buffer GC and 3% dimethyl sulfoxide (DMSO) were used forthe PCR reactions. The amplicons were assembled into a three-gene,two-promoter construct (“transporter insert”;P_(psaA)-tolC-P_(tsr2142)-acrAB) and placed in multiple cloning site ofrecombination vector pJB161 (SEQ ID # 15) to yield pJB1074. pJB161 (andpJB161-derived plasmids, including pJB1074) contain an upstream homologyregion (UHR) and a downstream homology region (DHR) that allowsrecombination into the pAQ7 plasmid of Synechococcus sp. PCC7002 at thelactate dehydrogenase locus (for pAQ7 plasmid sequence, see Genbank#CP000957). The homology regions flank a multiple cloning site (mcs),the natural terminator from the alcohol dehydrogenase gene fromZymomonas mobilis (adhII) and a kanamycin cassette which providesresistance in both E. coli and Synechococcus sp. PCC 7002. Thetransporter insert with flanking homology regions is provided as SEQ ID16.

Strain Construction. As described above, JCC803 is a strain ofSynechococcus sp. PCC 7002 that has been engineered to produce esters offatty acids (such as those found in biodiesel) when incubated in thepresence of alcohols. The strain contains a thioesterase (tesA), anacyl-CoA synthetase (fadD) and a wax synthase (wxs) inserted intoplasmid pAQ1 by homologous recombination.

The genes present in pJB161 and pJB1074 were integrated into the plasmidpAQ7 in Synechococcus sp. PCC 7002 (specifically, strain JCC803) usingthe following procedure. A 5 ml culture of JCC803 in A+ mediumcontaining 200 mg/L spectinomycin was incubated in an Infors shakingincubator at 150 rpm at 37° C. under 2% CO2/air and continuous light(70-130 μE m⁻² s⁻¹ PAR, measured with a LI-250A light meter (LI-COR))until it reached an OD730 of 1.14. For each plasmid, 500 μl of cultureand 5 μg of plasmid DNA were added into a microcentrifuge tube. Thetubes were then incubated at 37° C. in the dark rotating on a RotamixRKSVD (ATR, Inc.) on a setting of approximately 20. After 4 hours forpJB161 or 7 hours for pJB1074, the cells were pelleted using amicrocentrifuge. All but ˜100 μl of the supernatants were removed andthe cell pellets were resuspended using the remaining supernatant andplated on A+ agar plates. The plates were incubated overnight in aPercival lighted incubator under constant illumination (40-60 μE m⁻² s⁻¹PAR, measured with a LI-250A light meter (LI-COR)) at 37° C. for about24 hours. On the following day, spectinomycin and kanamycin solution wasadded underneath the agar of the plates to estimated concentration of 25mg/L spectinomycin and 50 mg/L kanamycin (assuming 40 ml A+ agar in theplate). These plates were placed back into the incubator until tinycolonies became visible. The plates were moved to another Percivalincubator under the same conditions except that 1% CO₂ was maintained inthe air (allows for faster growth). Approximately 110 colonies formedfor recombinant strains resulting from the pJB1074 transformation and2800 colonies resulting from the pJB160 transformation. A colony fromthe pJB161 transformation plate was designated JCC1132.

Thirty colonies were picked from the tolC-acrAB transformation plate andstreaked onto both an A+ plate with 100 mg/L spectinomycin and 0.05 mg/Lerythromycin and an A+ plate with 100 mg/L spectinomycin and 0.1 mg/Lerythromycin. Erythromycin is a substrate for the TolC-AcrAB transporter(Chollet et al. 2004) and served to verify function of the transporterin naturally erythromycin-sensitive Synechococcus sp. PCC 7002. Theplates were incubated in Percival lighted incubator at 37° C. underconstant illumination (40-60 μE m⁻² s⁻¹ PAR, measured with a LI-250Alight meter (LI-COR)) at 37° C. After two days, slight growth wasvisible on both plates. Eight days after streaking, variable growth andsurvival was evident on most of the streaks on the 0.05 mg/Lerythromycin plate. On the 0.1 mg/L erythromycin plate, all of thestreaks except for two had become nonviable. The same source coloniesthat produced the two viable streaks on 0.1 mg/L erythromycin producedstreaks that were healthy on the 0.05 mg/L erythromycin plate. One ofthese strains on the 0.1 mg/L erythromycin plate was designated JCC1585(see Table 13 for a list of strains).

TABLE 13 Strains and control strain investigated for the secretion ofbutyl esters. Parent Recombinant genes/ JCC # strain Promoters with lociMarker JCC1132 JCC803 pAQ1:: p_(trc)-tesa-fadd-wxs-aada; spectinomycinpAQ7::kan^(r) kanamycin JCC1585 JCC803 pAQ1::p_(trc)-tesa-fadd-wxs-aada; spectinomycin pAQ7::p_(psaa)-tolc-p_(tsr2142)-acrab- kanamycin kanr

Erythromycin Tolerance in Liquid Culture. To verify the improvedtolerance of JCC1585 to erythromycin compared to JCC1132, a 5 ml A+culture containing 200 mg/L spectinomycin and 0.5 mg/L erythromycin(JCC1585) or containing 200 mg/L spectinomycin and 50 mg/L kanamycin(JCC1132) were used to inoculate 30 ml of JB 2.1 containing 200 mg/Lspectinomycin and 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L erythromycin in 125ml culture flasks at an OD₇₃₀ of 0.1. These cultures were incubated inan Infors shaking incubator at 150 rpm at 37° C. under 2% CO₂/air andcontinuous light (70-130 μE m⁻² s⁻¹ PAR, measured with a LI-250A lightmeter (LI-COR)). Timepoints were taken at 5 and 10 days of growth,during which water loss was replaced through addition of milli-Q water.Table 14 shows OD₇₃₀ values of JCC1132 and JCC1585 cultures at day 5 and10 with different concentrations of erythromycin present in the medium.The JCC1585 cultures were tolerant of erythromycin concentrations of upto 1 mg/L (highest concentration tested) after 10 days while the JCC1132cultures had bleached under all concentrations of erythromycin tested.

TABLE 14 OD₇₃₀ Erythromycin Start OD₇₃₀ OD₇₃₀ Strain Concentration(mg/L) of Experiment Day 5 Day 10* JCC1132 0.5 0.1 5.72 — 0.6 0.1 4.76 —0.7 0.1 4.98 — 0.8 0.1 2.94 — 0.9 0.1 2.50 — 1.0 0.1 2.26 — JCC1585 0.50.1 6.60 7.34 0.6 0.1 6.34 6.20 0.7 0.1 5.82 5.74 0.8 0.1 5.80 4.84 0.90.1 5.34 5.04 1.0 0.1 5.58 5.12 *“—” indicates culture had bleached

To verify the improved tolerance of JCC1585 to erythromycin compared toJCC1132, a 5 ml A+ culture containing 200 mg/L spectinomycin and 0.5mg/L erythromycin (JCC1585) or containing 200 mg/L spectinomycin and 50mg/L kanamycin (JCC1132) were used to inoculate 30 ml of JB 2.1 mediacontaining 200 mg/L spectinomycin and 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/Lerythromycin in 125 ml culture flasks at an OD730 of 0.1. These cultureswere incubated in an Infors shaking incubator at 150 rpm at 37° C. under2% CO2/air and continuous light (70-130 μE m2/s PAR, measured with aLI-250A light meter (LI-COR)). Timepoints were taken at 5 and 10 days ofgrowth, during which water loss was replaced through addition of milli-Qwater. The JCC1585 cultures were tolerant of erythromycin concentrationsof up to 1 mg/L (highest concentration tested) after 10 days while theJCC1132 cultures had bleached under all concentrations of erythromycintested (Table 14).

Culture conditions. To test for secretion of butyl esters, 5 ml A+cultures with 200 mg/L spectinomycin and 50 mg/L kanamycin wereinoculated from colonies for JCC1132 and JCC1585. These cultures wereused to inoculate duplicate 30 ml cultures in JB2.1 medium containing200 mg/L spectinomycin and 50 mg/L kanamycin. At the beginning of theexperiment, 15 μl butanol (Sigma 34867) was added to each flask so thatfatty acid butyl esters (FABEs) would be produced by the cultures. Thesecultures were incubated in an Infors shaking incubator at 150 rpm at 37°C. under 2% CO₂/air and continuous light (70-130 μE m⁻² s⁻¹ PAR,measured with a LI-250A light meter (LI-COR)) for three days. At day 4of the experiment, 7.5 μl butanol was added to the cultures tocompensate for the experimentally determined stripping rate of butanolunder these conditions. Water loss through evaporation was replaced withthe addition of sterile Milli-Q water at day 7 and OD₇₃₀ readings weretaken for each culture.

Detection of Butyl Esters. An aliquot of 250 μl was removed from eachculture and centrifuged at 1500 rpm in Microcentrifuge 5424 (Eppendorf)for ˜2 min. The supernatants were removed and the pellets were suspendedin 500 μl milli-Q H₂O. The samples were centrifuged and the supernatantsdiscarded. An additional centrifugation step for 4 min was performed,and any remaining supernatant was removed. The weight of the tube andthe cell pellet were measured. One milliliter of acetone (Acros Organics326570010) containing 100 mg/L butylated hydroxytoluene (BHT,Sigma-Aldrich B1378) and 100 mg/L ethyl arachidate (Sigma A9010) wereadded to each pellet, and the mixture was pipetted up and down untilnone of the pellet remained on the wall of the tube. Each tube was thenvortexed for 15 s, and the weight of the tube, acetone solution, andcells was taken. The tubes were then spun down and 500 μl of supernatantwas submitted for GC analysis. From these samples, the percent dry cellweights of fatty acid butyl esters in the cell pellets were determined.

In order to quantify FABE's in the medium, 300 μL of a 20% (v/v) Span80(Fluka 85548) solution was added to each flask and mixed by swirling for30 seconds. These mixtures were then poured into 50 mL Falcon tubes.Five mL of isooctane containing 0.01% BHT and 0.005% ethyl arachidatewas added to the flasks and swirled for several seconds. The solutionswere then poured into the appropriate 50 mL Falcon tubes containing theculture from the flasks. The tube was then shaken for 10 seconds andcentrifuged using a Sorvall RC6 Plus superspeed centrifuge (ThermoElectron Corp) and a F13S14X50CY rotor (6000 rpm for 20 min). Onemilliliter of the organic phase (upper phase) was removed and submittedfor GC analysis.

The butyl esters produced by JCC803 and JCC803-derived strains wereidentified by GC/MS employing an Agilent 7890A GC/5975C EI-MS equippedwith a 7683 series autosampler. One microliter of each sample wasinjected into the GC inlet using a pulsed splitless injection (pressure:20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min)and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0mL/min. The GC oven temperature program was 50° C., hold one minute;10°/min increase to 280° C.; hold ten minutes. The GC/MS interface was290° C., and the MS range monitored was 25 to 600 amu. Butyl myristate[retention time (rt): 19.72 min], butyl palmitate (rt: 21.58 min) butylheptadecanoate (rt: 22.40 min), butyl oleate (rt: 23.04 min) and butylstearate (rt: 23.24 min) were identified by matching experimentallydetermined mass spectra associated with the peaks with mass spectralmatches found by searching in a NIST 08 MS database.

An Agilent 7890A GC/FID equipped with a 7683 series autosampler was usedto quantify the butyl esters. One microliter of each sample was injectedinto the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min,purge time: 0.2 min, purge flow: 15 mL/min), which was at a temperatureof 280° C. The column was an HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm), andthe carrier gas was helium at a flow of 1.0 mL/min. The GC oventemperature program was 50° C., hold one minute; 10°/min increase to280° C.; hold ten minutes. Butyl myristate (rt: 19.68 min], butylpalmitate (rt: 21.48 min), butyl heptadecanoate (rt: 22.32 min), butyloleate (rt: 22.95 min) and butyl stearate (rt: 23.14 min) werequantified by determining appropriate response factors for the number ofcarbons present in the butyl esters from commercially-available fattyacid ethyl esters (FAEEs) and FABEs. The calibration curves wereprepared for ethyl laurate (Sigma 61630), ethyl myristate (SigmaE39600), ethyl palmitate (Sigma P9009), ethyl oleate (Sigma 268011),ethyl stearate (Fluka 85690), butyl laurate (Sigma W220604) and butylstearate (Sigma S5001). The concentrations of the butyl esters presentin the extracts were determined and normalized to the concentration ofethyl arachidate (internal standard).

Peaks with areas greater than 0.05 could be integrated by theChemstation™ software (Agilent®), and the concentrations of the butylesters in both media and supernatant were determined from these values.The dry cell weight (DCW) of these strains was based on a measurement ofOD₇₃₀ and calculated based on the observed average DCW/OD relationshipof 0.29 g L⁻¹ OD⁻¹. In the case of the JCC1585 culture supernatant,small peaks for butyl myristate (flask 1 area: 1.26, flask 2: 2.23) andbutyl palmitate (flask 1 area: 5.16, flask 2: 5.62) were observed whileno peak with an area greater than 0.05 at these retention times wasfound in the media extraction of the JCC1132 cultures. The OD₇₃₀ percentdry cell weights of the FABEs in the cell pellets and the media aregiven in Table 15. The total % DCW of FABE's found in the cell pelletsis indicated, as is the % DCW of butyl myristate and butyl palmitatefound in the pellets and the media.

TABLE 15 Pellet butyl Media butyl myristate + myristate + FABEs butylpalmitate butyl palmitate Strain (flask) OD730 (% DCW) (% DCW) (% DCW)JCC1585 (1) 9.65 7.76 6.59 0.013 JCC1132 (1) 5.44 4.93 4.20 0 JCC1585(2) 8.50 7.79 6.65 0.018 JCC1132 (2) 4.48 4.60 3.85 0

Table 15 shows that the recombinant expression of tolC in an engineeredcyanobacterium provides for the secretion of a detectable fraction ofesters (in this case, butyl esters) synthesized by the engineered cell.The amount of secretion achieved can be modulated by increasingconcentrations of erythromycin or other transporter substrates, and/orthrough optimization of expression levels (promoter strength and codonoptimization strategies) and/or specifically targeting a cyanobacterialmembrane by employing appropriate cyanobacterial N-terminal leadersequences.

EXAMPLE 7 Secretion of Fatty Acids in Thermosynechococcus elongatus BP-1(Δaas)

Strain Construction. Thermosynechococcus elongatus BP-1long-chain-fatty-acid CoA ligase gene (aas, GenBank accession numberNP_(—)682091.1) was replaced with a thermostable kanamycin resistancemarker (kan_HTK, GenBank accession number AB121443.1) as follows:

Regions of homology flanking the BP-1 aas gene (Accession Number:NP_(—)682091.1) were amplified directly from BP-1 genomic DNA using theprimers in Table 16. PCR amplifications were performed with Phusion HighFidelity PCR Master Mix (New England BioLabs) and standard amplificationconditions.

TABLE 16 SEQ ID Restriction Primer Sequence NO: site added Upstream5′-GCTATGCCTGCAGGGGC 21 SbfI forward CTTTTATGAGGAGCGGTA-3′ Upstream5′-GCTATGGCGGCCGCTCTT 22 NotI reverse CATGACAGACCCTATGGATAC TA-3′ Down-5′-GCTATGGGCGCGCCTTAT 23 AscI stream CTGACTCCAGACGCAACA-3′ forward Down-5′-GCTATGGGCCGGCCGATC 24 FseI stream CTTGGATCAACTCACCCT-3′ reverse

The amplified upstream homologous region (UHR) was cloned into the UHRof a pJB5 expression vector containing kan_HTK by digesting the insertand vector individually with SbfI and NotI restriction endonucleases(New England BioLabs) following well known laboratory techniques.Digestions were isolated on 1% TAE agarose gel, purified using a GelExtraction Kit (Qiagen), and ligated with T4 DNA Ligase (New EnglandBioLabs) incubated at room temperature for 1 hour. The ligated productwas transformed into NEB 5-alpha chemically competent E. coli cells (NewEngland BioLabs) using standard techniques and confirmed by PCR. Thedownstream homologous region (DHR) was cloned into the resulting plasmidfollowing a similar protocol using AscI and FseI restrictionendonucleases (New England BioLabs). The final plasmid (pJB1349) waspurified using QIAprep Spin Miniprep kit (Qiagen) and the construct wasconfirmed by digestion with HindIII, AseI, and PstI restrictionendonucleases (New England BioLabs).

BP-1 was grown in 5 ml B-HEPES liquid media in a glass test tube (45°C., 120 rpm, 2% CO₂) to OD₇₃₀ 1.28. A 1 ml aliquot of culture wastransferred to a fresh tube and combined with 1 ug of purified pJB1349.The culture was incubated in the dark (45° C., 120 rpm, 2% CO₂) for 4hours. 4 ml of fresh B-HEPES liquid media were added and the culture wasincubated with light (45° C., 120 rpm, 2% CO₂) overnight. 500 μl of theresulting culture were plated in 3 ml of B-HEPES soft agar on B-HEPESplates containing 60 μg/ml kanamycin and placed in an illuminatedincubator (45° C., ambient CO₂) until colonies appeared (1 week), thenmoved into a 2% CO₂ illuminated incubator for an additional week.

Four randomly selected colonies (samples A-D) were independently grownin 5 ml B-HEPES liquid media with 60 μg/ml kanamycin in glass test tubes(45° C., 120 rpm, 2% CO₂) for one week. Replacement of aas gene wasconfirmed by PCR of whole cell genomic DNA by a culture PCR protocol asfollows. Briefly, 100 μl of each culture was resuspended in 50 μl lysisbuffer (96.8% diH₂O, 1% Triton X-100, 2% 1M Tris pH 8.5, 0.2% 1M EDTA).10 μl of each suspension were heated 10 min at 98° C. to lyse cells. 1μl of lysate was used in 15 μl standard PCR reactions using Quick-LoadTaq 2× Master Mix (New England BioLabs). The PCR product showed correctbands for an unsegregated knockout.

All cultures were maintained in fresh B-HEPES liquid media with 60 μg/mlkanamycin for an additional week. The PCR reaction described above wasrepeated, again showing correct bands for an unsegregated knockout.Cultures were maintained in liquid culture, and one representativeculture was saved as JCC1862.

Detection and quantification of free fatty acids in strains. Each of thefour independently inoculated cultures described above (samples A-D), aswell as BP-1, was analyzed for secretion of free fatty acids. OD₇₃₀ wasmeasured, and the volume in each culture tube was recorded. FreshB-HEPES liquid media was added to each tube to bring the total volume to5 ml and free fatty acids were extracted as follows:

Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/Lmethyl-β-cyclodextrin solution was added and samples were transferred to15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylatedhydroxytoluene in isooctane was added to each tube. Samples werevortexed 20 s, then centrifuged 5 min at 6000 RCF to fractionate. 500 μlof the isooctane layer were placed into a new tube and submitted for GCanalysis.

Concentrations of octanoic acid, decanoic acid, lauric acid, myristicacid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and1-nonadecene extractants were quantitated by gas chromatography/flameionization detection (GC/FID). Unknown peak areas in biological sampleswere converted to concentrations via linear calibration relationshipsdetermined between known authentic standard concentrations and theircorresponding GC-FID peak areas. Standards were obtained from Sigma.GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped witha 7683 series autosampler was used. 1 μl of each sample was injectedinto the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min,purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and thecarrier gas was helium at a flow of 1.0 ml/min. The GC oven temperatureprogram was 50° C., hold one minute; 10° C./min increase to 280° C.;hold ten minutes.

GC results showed that the unsegregated aas knockout increased fattyacid production relative to BP-1 (Table 17), with myristic and oleicacid making up the majority of the increase (Table 18).

TABLE 17 Fatty Acid Production by Sample Sample OD₇₃₀ Fatty acids (% DCWin media) Fatty acids (mg/L) A 6.25 0.20 3.66 B 5.20 0.11 1.71 C 5.600.24 3.85 D 5.80 0.23 3.83 BP-1 6.90 0.04 0.88

TABLE 18 Fatty Acid Production by Type Sample Myristic (mg/L) Palmitic(mg/L) Oleic (mg/L) A 0.119 0.051 0.032 B 0.000 0.072 0.042 C 0.1340.063 0.040 D 0.130 0.060 0.038 BP-1 0.000 0.044 0.000

EXAMPLE 8 Increased Production of Fatty Acids and Fatty Esters inThermosynechococcus elongatus BP-1 (Δaas)

Transformation of BP-1. As disclosed in PCT/US2010/042667, filed Jul.20, 2010, Thermosynechococcus elongatus BP-1 is transformed withintegration or expression plasmids using the following protocol. 400 mlThermosynechococcus elongatus BP-1 in B-HEPES medium is grown in a 2.8 lFernbach flask to an OD₇₃₀ of 1.0 in an Infors Multritron II shakingphotoincubator (55° C.; 3.5% CO₂; 150 rpm). For each transformation, 50ml cell culture is pelleted by centrifugation for 20 min (22° C.; 6000rpm). After removing the supernatant, the cell pellet is resuspended in500 μl B-HEPES and transferred to a 15 ml Falcon tube. To each 500 μlBP-1 cell suspension (OD₇₃₀ of ˜100), 25 μg undigested plasmid (or noDNA) is added. The cell-DNA suspension is incubated in a New Brunswickshaking incubator (45° C.; 250 rpm) in low light (˜3 μmol photons m⁻²s¹). Following this incubation, the cell-DNA suspension is made up to 1ml by addition of B-HEPES, mixed by gentle vortexing with 2.5 ml ofmolten B-HEPES 0.82% top agar solution equilibrated at 55° C., andspread out on the surface of a B-HEPES 1.5% agar plate (50 ml volume).Plates are left to sit at room temperature for 10 min to allowsolidification of the top agar, after which time plates are placed in aninverted position in a Percival photoincubator and left to incubate for24 hr (45° C.; 1% CO₂; 95% relative humidity) in low light (7-12 μmolphotons m⁻² s¹). After 24 hr, the plates are underlaid with 300 μl of 10mg/ml kanamycin so as to obtain a final kanamycin concentration of 60μg/ml following complete diffusion in the agar. Underlaid plates areplaced back in the Percival incubator and left to incubate (45° C.; 1%CO₂; 95% relative humidity; 7-12 μmol photons m⁻² s¹) for twelve days.

Increased Fatty Acids in BP-1. Thermosynechococcus elongatus BP-1 (Δaas)is first constructed as described in the above Example. BP-1 (Δaas) isshown to have elevated levels of both intracellular and extracellularlevels of free fatty acids relative to wild-type because mechanisticanalysis suggests that cells lacking an acyl-ACP synthetase have theinability to recycle exogenous or extracellular fatty acids; theextracellular fatty acid chains are diverted away from transport intothe inner cellular membrane while other transport systems are thought tocontinue to export fatty acids. Therefore, to up-regulate fatty acidproduction, BP-1 (Δaas) is transformed with a plasmid (e.g., pJB1349)carrying a thioesterase gene (see Table 3A). Increased cellular level offatty acid production may be attributed to the combination of the aasdeletion decreasing extracellular import of fatty acids and the additionof the thioesterase gene and/or thioesterase gene homologues.

Fatty Acid Esters. The thioesterase gene with or without the leadersequence removed (Genbank #NC 000913, ref: Chot and Cronan, 1993), theE. coli acyl-CoA synthetase fadD (Genbank #NC 000913, ref: Kameda andNunn, 1981) and the wax synthase (wxs) from Acinetobacter baylyi strainADPI (Genbank #AF529086.1, ref: Stóveken et al. 2005) genes are designedfor codon optimization, checking for secondary structure effects, andremoval of any unwanted restriction sites (NdeI, XhoI, BamHI, NgoMIV,NcoI, Sad, BsrGI, AvrII, BmtI, MiuI, EcoRI, SbfI, NotI, SpeI, XbaI, Pad,AscI, FseI). These genes are engineered into plasmid or integrationvectors (e.g., pJB1349) and assembled into a two gene operon (fadD-wxs)or a three gene operon (tesA-fadD-wxs) with flanking sites on theintegration vector corresponding to integration sites for transformationinto Thermosynechococcus elongatus BP-1. Integration sites include TS1,TS2, TS3 and TS4. A preferred integration site is the site of the aasgene. Host cells are cultured in the presence of small amounts ofethanol (1-10%) in the growth media under an appropriate promoter suchas Pnir for the production of fatty acid esters.

In another embodiment, Thermosynechococcus elongatus BP-1 host cell witha two gene operon (fadD-wxs) or a three gene operon (tesA-fadD-wxs) isengineered to have ethanol producing genes (PCT/US2009/035937, filedMar. 3, 2009; PCT/US2009/055949, filed Sep. 3, 2009; PCT/US2009/057694,filed Sep. 21, 2009) conferring the ability to produce fatty acidesters. In one plasmid construct, genes for ethanol production,including pyruvate decarboxylase from Zymomonas mobilis (pdc_(Zm)) andalcohol dehydrogenase from Moorella sp. HUC22-1 (adhA_(M)), areengineered into a plasmid and transformed into BP-1. In an alternateplasmid construct, the pyruvate decarboxylase from Zymobacter palmae(pdc_(Zp)) and alcohol dehydrogenase from Moorella sp. HUC22-1(adhA_(M)), are engineered into a plasmid and transformed into BP-1.These genes are engineered into plasmid or integration vectors (e.g.,pJB1349) with flanking sites on the integration vector corresponding tointegration sites for transformation into Thermosynechococcus elongatusBP-1. Integration sites include TS1, TS2, TS3 and TS4. A preferredintegration site is the site of the aas gene. In one configuration,expression of pdcZm and adhAM are driven by λ phage cI (“PcI”) and pEM7and in another expression strain driven by PcI and PtRNA^(Glu). In oneembodiment, a single promoter is used to control the expression of bothgenes. In another embodiment each gene expression is controlled byseparate promoters with PaphII or Pcpcb controlling one and PcIcontrolling the other.

EXAMPLE 9 Synechococcus sp. PCC 7002 (Δaas) with Various Thioesterases

Strain Construction. DNA sequences for thioesterase genes tesA, fatB,fatB1, and fatB2 were obtained from Genbank and were purchased from DNA2.0 following codon optimization, checking for secondary structureeffects, and removal of any unwanted restriction sites. Thioesterasegene fatB_mat is a modified form of fatB with its leader sequenceremoved.

TABLE 19 Thioesterase sources GenBank Gene name Organism origin proteinseq tesA Escherichia coli AAC73596 fatB Umbellularia californica(California Q41635 bay) fatB1 Cinnamomum camphora (camphor Q39473 tree)fatB2 Cuphea hookeriana AAC49269

The thioesterase genes were cloned into a pJB5 expression vectorcontaining upstream and downstream regions of homology to aquI(SYNPCC7002_A1189), pAQ3, and pAQ4 by digesting the inserts and vectorsindividually with AscI and NotI restriction endonucleases (New EnglandBioLabs) following known laboratory techniques. Digestions were isolatedon 1% TAE agarose gel, purified using a Gel Extraction Kit (Qiagen), andligated with T4 DNA Ligase (New England BioLabs) incubated at roomtemperature for one hour. The ligated product was transformed into NEB5-alpha chemically competent E. coli cells (New England BioLabs) usingstandard techniques. Purified plasmid was extracted using the QIAprepSpin Miniprep kit (Qiagen) and constructs were confirmed by PCR.

Synechococcus sp. PCC 7002 (Δaas) was grown in 5 ml A+ liquid media with25 μg/ml gentamicin in a glass test tube (37° C., 120 rpm, 2% CO₂) toOD₇₃₀ of 0.98-1.1. 500 μl of culture was combined with 1 μg purifiedplasmid in 1.5 ml microcentrifuge tubes and incubated in darkness 3-4hours. Samples were then plated on A+ agar plates with 3 or 6 mM ureaand incubated overnight 37° C. in the light. Selective antibiotics wereintroduced to the plates by placing stock solution spectinomycin underthe agar at a final concentration of 10 μg/mL, and incubating to allowdiffusion of the antibiotic. Plates were incubated at 37° C. with lightuntil plates cleared and individual colonies formed. Plates were thenmoved to an illuminated incubator at 2% CO₂. Cultures were maintained onliquid or agar A+ media containing 3-6 mM urea with 25 μg/ml gentamicin,100-200 μg/ml spectomycin, to promote plasmid segregation.

Thioesterase integration and attenuation was confirmed by PCR ofwhole-cell genomic DNA by a “culture PCR” protocol. Briefly, 100 μl ofeach culture was resuspended in 50 μl water or lysis buffer (96.8%diH₂O, 1% Triton X-100, 2% 1M tris pH 8.5, 0.2% 1M EDTA). 10 μl of eachsuspension were heated 10 min at 98° C. to lyse cells. 1 μl of lysatewas used in 10 μl standard PCR reactions using Quick-Load Taq 2× MasterMix (New England BioLabs) or Platinum PCR Supermix HiFi (Invitrogen).PCR products showed correct bands for segregated aquI, pAQ4 andunsegregated (pAQ3) integrants.

Detection and quantification of free fatty acids in strains. Individualcolonies were grown in A+ liquid media with 3 mM urea, 50 μg/mlgentamicin, 200 μg/ml spectomycin in glass test tubes (see Table 20).Cultures were maintained in liquid culture to promote segregation (37°C., 120 rpm, 2% CO₂). Liquid cultures were diluted to OD₇₃₀=0.2 in 5 mlA+ liquid media with 3 mM urea and no antibiotics in glass test tubesand incubated for seven days (37° C., 120 rpm, 2% CO₂). After one week,OD₇₃₀ was recorded and free fatty acids were extracted as follows:

Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/Lmethyl-β-cyclodextrin solution was added, and samples were transferredto 15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylatedhydroxytoluene in isooctane was added to each tube. Samples werevortexed 20 s and immediately centrifuged 5 min at 6000 RCF tofractionate. 500 μl of the isooctane layer were sub-sampled into a newtube and submitted for GC analysis.

Concentrations of octanoic acid, decanoic acid, lauric acid, myristicacid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and1-nonadecene extractants were quantitated by gas chromatography/flameionization detection (GC/FID). Unknown peak areas in biological sampleswere converted to concentrations via linear calibration relationshipsdetermined between known authentic standard concentrations and theircorresponding GC-FID peak areas. Standards were obtained from Sigma.GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped witha 7683 series autosampler was used. 1 μl of each sample was injectedinto the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min,purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and thecarrier gas was helium at a flow of 1.0 ml/min. The GC oven temperatureprogram was 50° C., hold one minute; 10° C./min increase to 280° C.;hold ten minutes.

GC results showed increased fatty acid secretion in the thioesterasestrains relative to Synechococcus sp. PCC 7002 JCC138 (Table 20). Thespecific enrichment profile of each culture was thioesterase dependent(Table 21).

TABLE 20 Fatty acid secretion in tesA, fatB_mat strains Fatty Acids (%DCW Fatty acids Sample Location Promoter Thioesterase Δaas OD₇₃₀ inmedia) (mg/ml) JCC 138 — — — — 11.80 0.11 3.81 JCC 1648 pAQ4 P(nir07)tesA yes 5.56 2.76 44.45 JCC 1751 pAQ3 P(nir07) tesA yes 7.68 2.29 51.10JCC 1755 pAQ3 P(nir07) fatB_mat yes 3.92 1.79 20.38

TABLE 21 Fatty acids by type % DCW of compounds Sample Lauric MyristicPalmitoleic Palmitic Oleic Stearic JCC 138 0.000 0.061 0.000 0.000 0.0000.050 JCC1648 0.342 1.557 0.238 0.000 0.260 0.360 JCC 1751 0.146 0.5390.165 1.145 0.158 0.143 JCC1755 0.940 0.224 0.289 0.143 0.197 0.000

Individual colonies of JCC1704, JCC1705, and JCC1706 were grown forthree days in A+ liquid media with 3 mM urea, 25 μg/ml gentamicin, 100μg/ml spectomycin in glass test tubes (37° C., 120 rpm, 2% CO₂).Cultures were diluted to OD₇₃₀=0.2 in 5 ml A+ liquid media with 3 mMurea and no antibiotics in glass test tubes and incubated at 37° C., 120rpm, 2% CO₂. After 11 days, OD₇₃₀ was recorded and free fatty acids wereextracted as follows:

Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/Lmethyl-β-cyclodextrin solution was added and samples were transferred to15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylatedhydroxytoluene in isooctane was added to each tube. Samples werevortexed 20 s and immediately centrifuged 5 min at 6000 RCF tofractionate. 500 μl of the isooctane layer were sub-sampled into a newtube and submitted for GC analysis.

Concentrations of octanoic acid, decanoic acid, lauric acid, myristicacid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and1-nonadecene extractants were quantitated by gas chromatography/flameionization detection (GC/FID). Unknown peak areas in biological sampleswere converted to concentrations via linear calibration relationshipsdetermined between known authentic standard concentrations and theircorresponding GC-FID peak areas. Standards were obtained from Sigma.GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped witha 7683 series autosampler was used. 1 μl of each sample was injectedinto the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min,purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and thecarrier gas was helium at a flow of 1.0 ml/min The GC oven temperatureprogram was 50° C., hold one minute; 10° C./min increase to 280° C.;hold ten minutes.

GC results showed increased fatty acid secretion relative to JCC138 butto a lesser degree than tesA or fatB_mat (Table 22). The specificenrichment profile of each culture was thioesterase dependent (Table23).

TABLE 22 Fatty acid secretion in fatB, fatB1, fatB2 strains Fatty AcidsFatty (% DCW acids Sample Location Promoter Thioesterase Δaas OD₇₃₀ inmedia) (mg/ml) JCC 1648 pAQ4 P(nir07) tesA yes 11.2 6.66 216.283 JCC1648 pAQ4 P(nir07) tesA yes 11.6 5.74 193.236 JCC 1704 aquI P(nir07)fatB yes 15.80 0.39 17.72 JCC 1704 aquI P(nir07) fatB yes 16.80 0.4019.56 JCC 1705 aquI P(nir07) fatB1 yes 15.6 0.42 19.19 JCC 1705 aquIP(nir07) fatB1 yes 16.3 0.43 20.44 JCC 1706 aquI P(nir07) fatB2 yes 17.50.40 20.25 JCC 1706 aquI P(nir07) fatB2 yes 16.5 0.41 19.86

TABLE 23 Fatty acids by type % DCW of compounds Sample Lauric MyristicPalmitoleic Palmitic Oleic Stearic JCC 1648 0.233 1.408 0.264 3.9190.223 0.611 JCC 1648 0.201 1.196 0.183 3.564 0.131 0.470 JCC 1704 0.0000.057 0.107 0.073 0.087 0.063 JCC 1704 0.000 0.062 0.113 0.073 0.0940.060 JCC 1705 0.000 0.058 0.110 0.089 0.099 0.068 JCC 1705 0.000 0.0580.107 0.092 0.101 0.074 JCC 1706 0.000 0.054 0.098 0.090 0.085 0.071 JCC1706 0.000 0.056 0.106 0.086 0.100 0.068

EXAMPLE 10 Fatty Acid Production Under Inducible or Repressible System

Construction of the promoter-uidA expression plasmid. The E. coli uidAgene (Genbank AAB30197) was synthesized by DNA 2.0 (Menlo Park, Calif.),and was subcloned into pJB5. The DNA sequences of theammonia-repressible nitrate reductase promoters P(nirA) (SEQ ID NO:17),P(nir07) (SEQ ID NO:18), and P(nir09) (SEQ ID NO:19) were obtained fromGenbank. The nickel-inducible P(nrsB) promoter (SEQ ID NO:20), nrsS andnrsR were amplified from Synechocystis sp. PCC 6803. The promoters werecloned between NotI and NdeI sites immediately upstream of uidA, whichis flanked by NdeI and EcoRI.

In addition, plasmids containing two 750-bp regions of homology designedto remove the native aquI (A1189) or the ldh (G0164) gene fromSynechococcus sp. PCC 7002 were obtained by contract synthesis from DNA2.0 (Menlo Park, Calif.). Using these vectors, 4 constructs wereengineered and tested for GUS activity. Final transformation constructsare listed in Table 24. All restriction and ligation enzymes wereobtained from New England Biolabs (Ipswich, Mass.). Ligated constructswere transformed into NEB 5-α competent E. coli (High Efficiency) (NewEngland Biolabs: Ipswich, Mass.).

TABLE 24 Genotypes of JCC138 transformants Insert location PromoterMarker ldh P(nirA) kanamycin aquI P(nir07) spectinomycin aquI P(nir09)spectinomycin ldh P(nrsB) kanamycin

Plasmid transformation into JCC138. The constructs as described abovewere integrated onto either the genome or pAQ7 of JCC138, both of whichare maintained at approximately 7 copies per cell. The followingprotocol was used for integrating the DNA cassettes. JCC138 was grown inan incubated shaker flask at 37° C. at 1% CO₂ to an OD₇₃₀ of 0.8 in A⁺medium. 500 μl of culture was added to a microcentrifuge tube with 1 μgof DNA. DNA was prepared using a Qiagen Qiaprep Spin Miniprep Kit(Valencia, Calif.) for each construct. Cells were incubated in the darkfor one hour at 37° C. The entire volume of cells was plated on A⁺plates with 1.5% agar supplemented with 3 mM urea when necessary andgrown at 37° C. in an illuminated incubator (40-60 μE/m2/s PAR, measuredwith a LI-250A light meter (LI-COR)) for approximately 24 hours. 25μg/mL of spectinomycin or 50 μg/mL of kanamycin was introduced to theplates by placing the stock solution of antibiotic under the agar, andallowing it to diffuse up through the agar. After further incubation,resistant colonies became visible in 6 days. One colony from each platewas restreaked onto A⁺ plates with 1.5% agar supplemented with 6 mM ureawhen necessary and 200 μg/mL spectinomycin or 50 μg/mL of kanamycin.

Measurement of GUS activity. The GUS (beta-glucuronidase) reportersystem was used to test the inducibility or repressibility of severalpromoters. This system measures the activity of beta-glucuronidase, anenzyme from E. coli that transforms colorless or non-fluorescentsubstrates into colored or fluorescent products. In this case, MUG(4-methylumbelliferyl β-D-glucuronide) is the substrate, and ishydrolyzed by beta-glucuronidase to produce the florescent product MU(4-methylumbelliferone), which is subsequently detected and quantifiedwith a fluorescent spectrophotometer.

Strains containing uidA constructs under urea repression were incubatedto OD₇₃₀ between 1.8 and 4. These cells were subcultured to OD₇₃₀ 0.2 in5 mL A+ media supplemented with 0, 3, 6, or 12 mM urea plus either 100μg/mL spectinomycin or 50 μg/ml kanamycin and incubated for 24 hours.JCC138 was cultured in 5 mL A+ media for 24 hours. The strain containinggus under nickel-inducible expression was cultured for 3 days, thensubcultured to OD₇₃₀ 0.2 in 5 mL A+ supplemented with 0, 2, 4, or 8 MNiSO₄. These cells were incubated for 6 hours. To harvest cells,cultures were spun for 5 minute at 6000 rpm. Pellets were resuspended in1 mL 1×GUS extraction buffer (1 mM EDTA, 5.6 mM 2-mercaptoethanol, 0.1 Msodium phosphate, pH 7) and lysed with microtip sonication pulsing 0.5seconds on and 0.5 seconds off for 2 min. Total protein was analyzedwith Bio-Rad (Hercules, Calif.) Quick Start Bradford assay, and extractswere subsequently analyzed for GUS activity using a Sigma (St Louis,Mo.) β-Glucuronidase Fluorescent Activity Detection Kit. Relativeactivities of the 4 promoters are found in Table 25.

TABLE 25 GUS activities of inducible/repressible promoters promoter mMurea uM NiSO₄ (ABS/mg × 10⁶) P(nirA) 0 — 121.9 3 — 8 6 — 11.62 12 — 7.81P(nir07) 0 — 396.39 3 — 23.61 6 — 30.89 12 — 33.13 P(nir09) 0 — 97.77 3— 12.47 6 — 12.35 12 — 12.1 P(nrsB) — 0 24.97 — 2 286.96 — 4 257.26 — 8423.77 no uidA gene — — 6.4

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. Allpublications, patents and other references mentioned herein are herebyincorporated by reference in their entirety.

REFERENCES

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1. A method for producing fatty acid esters, comprising: (i) culturingan engineered cyanobacterium in a culture medium, wherein saidengineered cyanobacterium comprises a recombinant acyl-CoA synthetase(EC 6.2.1.3) and a recombinant wax synthase (EC 2.3.1.75); and (ii)exposing said engineered cyanobacterium to light and carbon dioxide,wherein said exposure results in the incorporation of an alcohol intofatty acid esters produced by said engineered cyanobacterium, wherein atleast one of said fatty acid esters is selected from the groupconsisting of a tetradecanoic acid ester, a Δ9-hexadecenoic acid ester,a hexadecanoic acid ester, a heptadecanoic acid ester, a Δ9-octadecenoicacid ester, and an octadecanoic acid ester, wherein the amount of saidfatty acid esters produced by said engineered cyanobacterium isincreased relative to the amount of fatty acid produced by an otherwiseidentical cell lacking said recombinant acyl-CoA synthetase orrecombinant wax synthase.
 2. The method of claim 1, wherein saidengineered cyanobacterium further comprises a recombinant thioesterase.3. The method of claim 1, wherein said alcohol is an exogenously addedalcohol selected from the group consisting of methanol, ethanol,propanol, isopropanol, butanol, hexanol, cyclohexanol, and isoamylalcohol.
 4. The method of claim 3, wherein said esters include ahexadecanoic acid ester and an octadecanoic acid ester.
 5. The method ofclaim 4, wherein the amount of hexadecanoic acid ester produced isbetween 1.5 and 10 fold greater than the amount of octadecanoic acidester.
 6. The method of claim 4, wherein at least 50% of the estersproduced by said engineered cyanobacterium are hexadecanoic acid esters.7. The method of claim 4, wherein said alcohol is butanol.
 8. The methodof claim 7, wherein the yield of fatty acid butyl esters is at least 5%dry cell weight.
 9. The method of claim 4, wherein said alcohol isethanol.
 10. The method of claim 9, wherein the yield of ethyl esters isat least 1% dry cell weight.
 11. The method of claim 4, wherein saidalcohol is methanol.
 12. The method of claim 1, wherein said engineeredcyanobacterium further comprises a deletion or knock-out of anendogenous gene encoding a long-chain-fatty-acid ACP ligase.
 13. Themethod of claim 1, wherein said engineered cyanobacterium is athermophilic cyanobacterium.